Virology Final Flashcards

Question

Describe viral entry in the replication cycle?

Answer 1

The Baltimore classification system categorizes viruses into seven groups based on their type of nucleic acid and their replication strategy. The groups include: DNA viruses (single-stranded and double-stranded), RNA viruses (single-stranded and double-stranded), and retroviruses, which use reverse transcription to convert their RNA genome into DNA. This system helps in understanding the biology of viruses and their interactions with host cells.

Answer 2

Capsid Nucleocapsid Envelope

Answer 3

Genetic economy refers to the principle that organisms optimize the use of their genetic resources to maximize efficiency and minimize unnecessary genetic material. This concept often involves self-assembly, where proteins and other molecules spontaneously organize into functional structures without the need for additional energy input. By utilizing genetic economy, organisms can effectively produce complex biomolecules while conserving resources and energy.

Answer 4

Cubic Icosahedral

Answer 5

- Small, non-enveloped virus that primarily infects animals, including humans. - Notable example: canine parvovirus. - Contains a single-stranded DNA genome. - Features an icosahedral protein capsid. - Capsid proteins exhibit a jelly-roll B barrel fold, providing stability and protection for the viral genome. - Highly resilient in the environment. - Can cause diseases, particularly in young or immunocompromised animals.

Answer 6

quasi-equivalent

Answer 7

Protein subunits can move slightly to accommodate distortions therefore they have built in flexibility

Answer 8

- Contain Lipid bilayers that have a similar protein composition as the cellular membrane from which they were derived. So say it buds off from ER, it will have a similar composition to that of the ER.

Answer 9

Flaviviruses bud at the endoplasmic reticulum (ER) of host cells, acquiring their lipid envelope during this process. After budding, they are transported through the Golgi apparatus and eventually released from the cell. They can cause diseases ranging from mild fever to severe conditions like encephalitis or haemorrhagic fever

Answer 10

The ER-Golgi intermediate compartment (ERGIC) acts as a key trafficking station between the endoplasmic reticulum (ER) and the Golgi apparatus, serving as an interface for vesicle transport in eukaryotic cells. During viral budding, particularly in viruses like coronaviruses, the ERGIC functions as the primary site where newly assembled viral nucleocapsids acquire their lipid envelope. The ERGIC’s membranes, rich in host lipids, provide the essential lipid bilayer needed to form the viral envelope, while simultaneously ensuring the proper incorporation of viral spike or glycoproteins into the budding virions. Viral glycoproteins, synthesised in the ER, are transported to the ERGIC, where they are embedded in the membrane, allowing the virions to adopt the necessary surface proteins for host cell recognition and entry. The ERGIC’s central role in vesicle trafficking ensures that viral particles are sorted and processed efficiently, directing them toward the Golgi apparatus for further maturation. This pathway allows the virus to be packaged and secreted properly, with a fully intact and functional envelope, which is crucial for its stability and infectivity upon release into the extracellular environment

Answer 11

- Most viruses will have Viral glycoproteins (proteins with a sugar attached) embedded in the lipid bilayer. Can be any sort of shape

Answer 12

1. Large glycosylated (the controlled enzymatic modification of an organic molecule, especially a protein, by addition of a sugar molecule) external domain (ectodomain) 2. Hydrophobic transmembrane anchor domain 3. Short internal cytoplasmic tail - Envelope proteins are synthesised on ribosomes in the ER → inserted in plasma membrane via standard export pathways for cell-surface proteins - Glycosylation occur in ER/Golgi

Answer 13

- Has HA (Hemagglutinin) which forms a trimer that binds to cell receptors and mediates fusion between viral envelope and cell membrane - The transmembrane anchor domain are alpha helices that span 3 nm thick hydrophobic part of lipid bilayer - Tail faces cytoplasm before a virus buds off * Can interact with various proteins - Glycosylation of external domain: * Prevents dehydration of the external surfaces of virus particles * Reduces protein-protein interactions to prevent aggregation

Answer 14

- N-terminus orientation: Type I integral membrane proteins have their N-terminus facing the extracellular side and their C-terminus on the cytoplasmic side. - Transmembrane domain: They possess a single hydrophobic transmembrane domain, usually an alpha-helix, that anchors them in the lipid bilayer. - Signal sequence: A cleavable signal sequence at the N-terminus directs these proteins to the endoplasmic reticulum (ER) during synthesis. - Functions: These proteins play roles in signalling, transport, and cell-cell interactions, with examples like hormone receptors (e.g., insulin receptor) and immune cell surface proteins (e.g., CD markers).

Answer 15

- C-terminus orientation: Type II integral membrane proteins have their N-terminus facing the cytoplasmic side and their C-terminus on the extracellular side. - Transmembrane domain: They contain a single hydrophobic transmembrane domain that anchors them in the lipid bilayer, typically as an alpha-helix. - Signal-anchor sequence: These proteins use an internal signal-anchor sequence, which acts both as a signal for targeting to the ER and as the membrane-anchoring region. - Functions: They are involved in processes like enzymatic activity, signalling, and cell recognition, with examples including transferrin receptors and certain viral glycoproteins.

Answer 16

- Type I membrane proteins have a N-terminal signal sequence that is cleaved by a peptidase when inserted in the ER during synthesis - Type II use the transmembrane anchor as the signal sequence - Signal sequence is an amino acid that directs thing on where to go

Answer 17

Budding is a key mechanism by which viruses acquire their envelopes during the replication process. In this process, newly formed viral particles associate with the host cell's membrane, where viral glycoproteins are embedded. The viral particle pushes against the membrane, causing the membrane to wrap around it and eventually pinch off, resulting in the release of an enveloped virus. This method allows the virus to obtain a portion of the host cell membrane, which helps in evading the host immune response and aids in the virus's ability to infect new cells

Answer 18

- Genome packaged within capsid - In some viruses, capsids assemble around the genome, and in some the genome is inserted into a preformed shell

Answer 19

Some viral capsids may require scaffolding proteins which organise and stabilise the interactions between different signalling molecules, creating a framework that enhances the efficiency and specificity of cellular signalling pathways. By bringing together various enzymes, receptors, and other proteins, they ensure precise spatial and temporal coordination of biological processes. * Assist with formation of a procapsid, which is a precursor to a full capsid which contains no DNA * Not included in the mature virion

Answer 20

A procapsid is an intermediate structure formed during the assembly of a virus, specifically a protein shell that encases the viral genome. It is typically composed of structural proteins that will later mature into a fully functional capsid, often undergoing conformational changes. Procapsids serve as a scaffold for the packaging of the viral nucleic acid and are crucial in the viral life cycle.

Answer 21

concatemer

Answer 22

Nucleocapsids

Answer 23

Matrix proteins play a crucial role in viral budding by forming a layer between the viral envelope and the nucleocapsid, often serving as connecting bridges that stabilize the structure. In many helical nucleocapsids, these proteins are encoded by the virus and are located just beneath the envelope. For retroviruses like HIV-1, the assembly of nucleocapsids occurs directly at the membrane during budding, where a precursor protein (gag protein) is cleaved to produce both matrix and nucleocapsid proteins, facilitating efficient release of the virus from host cells.

Answer 24

Budding can be driven by envelope glycoproteins, which are essential for forming viral envelopes during release from host cells. Some viruses can generate empty envelopes—membranes without nucleocapsids—where glycoproteins facilitate this process. In certain retroviruses that produce "bald" particles (nucleocapsids wrapped in membranes without envelope proteins), the genes for glycoproteins may be lost, but a layer of gag proteins on the inner plasma membrane interacts with the lipid bilayer to promote budding. The final pinch-off often requires cellular proteins, such as ESCRT (endosomal sorting complexes required for transport), for efficient release.

Answer 25

Virion disassembly occurs when viruses release their genomes upon entering host cells. This release can happen through various mechanisms, with proteolytic cleavage of capsid proteins being the most common, along with self-cleavage, pH-dependent cleavage, and membrane fusion. The process often involves unspooling the viral genome into the cell and interacting with cytoplasmic components, as virions are energetically metastable and can easily dissociate when triggered by binding to cell surface receptors. Notably, assembly and disassembly are distinct processes, not merely reversals of each other.

Answer 26

- Type of nucleic acid genome (DNA or RNA) * Strandedness of nucleic acids (single- or double-stranded) * Topology of nucleic acids (linear, circular, fragmented) * Capsid symmetry (icosahedral, helical, none) * Presence or absence of an envelope

Answer 27

- Bacteria * Archaea * Lower eukaryotes (fungi, protozoa, algae) * Plants * Invertebrates * Vertebrates (including humans)

Answer 28

Viruses with ssDNA genomes tend to be small and have few genes with ssDNA being susceptible to degradation * Viruses with dsDNA genomes include some of the largest known viruses * E.g. most bacteriophages have dsDNA genomes * Most plant viruses and some vertebrate viruses have +ve strand RNA genomes * Most known viruses with -ve strand RNA genomes have helical nucleocapsids * Most viruses with dsRNA genomes have a segmented genome and capsids with icosahedral symmetry (mostly infect fungi) * Viruses with a reverse transcriptase (RT) step in their replication cycle can have RNA or DNA genomes.

Answer 29

- Viruses that possess reverse transcriptase (RT) in their replication cycle package this enzyme within their virions. This allows them to convert their RNA genomes into DNA after entering the host cell. Notable virus families that utilize reverse transcriptase include Retroviridae, Hepadnaviridae, and Belpaoviridae. Additionally, negative-strand RNA viruses and double-stranded RNA (dsRNA) viruses also package RNA-dependent RNA polymerase (RdRp) within their virions, which is crucial for synthesising complementary RNA strands from their viral RNA genomes.

Answer 30

- Self-replicating RNAs → DNA likely involved: 1) RNA-dependent RNA polymerases (RNA replication) 2) RNA-dependent DNA polymerases (reverse transcriptases) (RNA → DNA) 3) DNA-dependent RNA polymerases (DNA → mRNA) 4) DNA-dependent DNA polymerases (DNA replication) * Small and medium-sized DNA viruses could have arisen as independently replicating genetic elements in cells * Large DNA viruses could have evolved from cellular forms that became obligatory intracellular parasites

Answer 31

* Structural proteins * Proteins that stimulate DNA replication enzymes in host * Proteins that recognize and bind to viral DNA and assemble cellular replication machinery

Answer 32

* Group 1: ssDNA genomes (small, without envelopes) * Group 2: dsDNA genomes (widespread, can be very large) * Group 3: + strand RNA genomes (most common in plants, also found in vertebrates) * Group 4: - strand RNA genomes (major infectious diseases in humans, helical nucleocapsids) * Group 5: dsRNA genomes (mostly in fungi, segmented genomes) * Group 6: Viruses that use reverse transcriptase (can be DNA or RNA) * Group 7: Satellite viruses, satellite nucleic acids, and viroids (very small genomes, highly dependent on host cells and helper viruses for packaging or replication)

Answer 33

Enveloped viruses can enter by: * Fusion and fission of the envelope with the plasma membrane * Receptor-mediated endocytosis followed by fusion/fission with an endosome

Answer 34

Filopedia act almost as arms on the corona virions. Filopodia can directly transport the virus to endocytic hot spots to avoid the virus from disorderly searching on the plasma membrane

Answer 35

- Attachment factor(s) / Adhesion receptors: Cell surface component(s) involved in binding of virion to cell but not uptake. (Usually considered primary receptors) * E.g., carbohydrates moieties on glycoproteins, proteoglycans, glycolipids etc. - Entry Receptors play an active role e.g. conformational changes, cell signalling endocytosis etc. (Usually considered secondary or co-receptors)

Answer 36

Host cell receptors interact with surface components of a virion by specifically binding to viral proteins or glycoproteins on the virion's outer surface, facilitating the initial attachment. This interaction is highly selective, as the virus's surface components are shaped to fit particular receptors on the host cell, like a "lock-and-key" mechanism. Once the virion attaches to the receptor, this binding often triggers conformational changes in the viral structure or host membrane, leading to the fusion of the viral envelope with the host cell membrane or receptor-mediated endocytosis, allowing the virus to enter the cell and initiate infection.

Answer 37

Multiple weak interactions can work together to create a strong overall binding. This is often due to multivalency, where several interactions occur simultaneously, increasing the strength and stability of the attachment, even if each individual interaction is weak on its own. This cumulative effect strengthens the overall connection between molecules or cells.

Answer 38

- Some viruses enter by macropinocytosis - transient ruffling of plasma membrane and internalization of fluid, solutes, membranes and small particles attached to plasma membrane → generally non-specific. - Internalization forms fairly large vacuoles called macropinosomes - Viruses can trigger macropinocytosis by exploiting cell signalling (fairly complex, see next slide)

Answer 39

Fusion proteins are generally synthesized, folded and assembled in ER → must undergo maturation steps e.g.: * Cleaved while transiting ER-Golgi to plasma membrane by cellular proteases (e.g. influenza) * Cleaved by host cell proteases once bound to target cell * Conformational changes that reveal a fusion peptide – short hydrophobic region within a viral envelope protein → gets inserted into target cellular membrane during virus-induced membrane fusion. * Fusion proteins play a critical role in viral infection → can be therapeutic targets

Answer 40

This is a picornovirus btw, notice the structural proteins of the virus

Answer 41

lysosomaltropic agents chemotropic agents Lysosomotropic agents are compounds that preferentially accumulate within lysosomes due to their physicochemical properties, such as being weakly basic and lipophilic. These agents are often protonated in the acidic environment of lysosomes, leading to their retention. Lysosomotropic agents can disrupt lysosomal function, influence intracellular trafficking, or induce lysosomal membrane permeabilisation, which may trigger cell death or autophagy. Examples include chloroquine and hydroxychloroquine, which are used in research and therapy for their effects on lysosomal pH and activity. Chemotropic agents are substances that direct the movement or growth of cells, tissues, or organisms towards or away from specific chemical stimuli, a process called chemotropism. These agents play a key role in biological processes like neuronal pathfinding, angiogenesis, and fungal growth. For example, nerve growth factor (NGF) acts as a chemotropic agent in guiding axonal growth, while VEGF (vascular endothelial growth factor) directs endothelial cells during blood vessel formation

Answer 42

The N protein (nucleocapsid protein) binds to the viral RNA genome, wrapping it into a ribonucleoprotein complex that shields the RNA from degradation and allows it to maintain a stable helical structure within the virion. This stabilisation is crucial for the virus’s assembly and protects the RNA during the infection process, supporting efficient replication once inside a host cell. By directly interacting with the M protein at the host cell’s membrane, the N protein facilitates the assembly of the virion, helping to package the RNA genome into new viral particles. Additionally, the N protein regulates viral RNA synthesis and modulates host immune defences, notably by interfering with interferon production, which aids in evading immune detection.

Answer 43

icosahedral symmetry helical

Answer 44

Coronavirus spike (S) proteins are large, trimeric proteins that protrude from the virus surface, giving it a crown-like appearance. These proteins are responsible for mediating entry into host cells by binding to specific receptors, such as ACE2 in SARS-CoV-2, on the host cell surface. The spike protein has two main subunits

Answer 45

This subunit is primarily responsible for recognising and binding to the host cell. It contains the receptor-binding domain (RBD), a highly variable region that allows the virus to target specific receptors on host cells (such as ACE2 in SARS-CoV-2). When the S1 subunit binds to the receptor, it triggers a series of structural changes in the spike protein, preparing it for membrane fusion. The S1 subunit is also a major target for neutralising antibodies, as blocking this interaction can prevent viral entry.

Answer 46

After S1 binds to the host receptor, the S2 subunit facilitates fusion between the viral and host cell membranes. It contains several key regions, including the fusion peptide, heptad repeats, and transmembrane domain, which enable the fusion process. Following receptor engagement, S2 undergoes conformational changes that expose the fusion peptide, which inserts into the host cell membrane. This action pulls the membranes together, allowing the viral genome to enter the host cell.

Answer 47

- Spike protein generally mediates entry via fusion * External S1 subunit mediates attachment * Stalk subunit S2 (a class I fusion protein) facilitates fusion * Series of conformational changes → insertion of S2 into target cell membrane → brings cell membrane and viral envelope into close contact - Some CoV S proteins can also cause formation of syncytia * In some cases, fusion can be pH dependent (e.g., fusion induced at low pH → indicates entry via endosomes, followed by fusion with endosomal membranes) - Fusion at plasma membrane is also required for egress from cells

Answer 48

TMPRSS2: This cell surface serine protease is located on the host cell membrane and activates the spike (S) protein through cleavage. TMPRSS2 cuts the spike protein at specific sites, which triggers conformational changes in the S2 subunit that are necessary for fusion between the viral and host cell membranes. This activation pathway allows the virus to enter the host cell directly at the plasma membrane, bypassing endosomal entry, which can lead to a quicker fusion process and viral replication

Answer 49

Cathepsin L: Found within the endosomes, cathepsin L is a lysosomal protease that activates the spike protein when the virus enters the cell via endocytosis. After the virus is engulfed into an endosome, the acidic environment activates cathepsin L, which cleaves the spike protein, again enabling the conformational changes in S2 needed for fusion with the endosomal membrane. This pathway provides an alternative route for viral entry if TMPRSS2 is not present or accessible on the cell surface.

Answer 50

The replicase gene (Gene 1) in coronaviruses is a large, essential gene that encodes proteins responsible for viral replication and transcription. This gene occupies approximately two-thirds of the viral genome and is transcribed and translated early in infection. It encodes two overlapping open reading frames, ORF1a and ORF1b, which are translated into polyproteins pp1a and pp1ab.

Answer 51

ribosome frameshifts during the translation of ORF1a are a well-orchestrated mechanism in certain viruses that allows for the efficient production of viral polyproteins. The ribosome shifts frames at a slippery sequence near the junction of ORF1a and ORF1b, facilitated by a secondary RNA structure such as a pseudoknot. This frameshift is necessary for the virus to maximize the usage of its genome and produce the necessary proteins for replication.

Answer 52

pp1a gets broken down into multiple 10–11 mature proteins, including viral proteases (like the main protease and papain-like protease) which are crucial for processing both pp1a and pp1ab into functional nsps. These nsps have various roles, including modifying the host environment to support viral replication, initiating RNA synthesis, and subverting host immune responses.

Answer 53

pp1ab, produced when a ribosomal frameshift allows translation of the ORF1b region, yields up to 16 mature proteins. The additional nsps produced from ORF1b include key replication enzymes like the RNA-dependent RNA polymerase (RdRp), helicase, and exonuclease, which are vital for accurate viral genome replication and proofreading.

Answer 54

The high conservation of ORF1b across coronaviruses suggests its importance in maintaining essential replication functions, indicating that mutations in this region are less tolerated. This conservation highlights the critical nature of ORF1b’s encoded proteins for viral survival, as they are responsible for core processes in replication and genomic integrity.

Answer 55

The papain-like cysteine proteinases (PLP1 and PLP2), derived from nsp3, are responsible for cleaving specific sites within pp1a and pp1ab. These proteases not only help to release individual functional non-structural proteins (nsps) but also play a role in evading the host immune response by interfering with host cell pathways.

Answer 56

RNA-dependent RNA polymerase (RdRp), encoded by ORF1b as part of nsp12, is vital for coronavirus replication and transcription: Viral RNA Replication: RdRp synthesises complementary RNA strands from the viral genome, producing full-length copies for new virions and subgenomic RNAs for protein synthesis. Subgenomic RNA Synthesis: It generates subgenomic mRNAs necessary for producing various viral proteins, allowing efficient use of the viral genome. Proofreading Activity: RdRp has proofreading capability, helping maintain genomic integrity by reducing mutation rates during replication

Answer 57

RNA Helicase: This enzyme unwinds RNA structures, facilitating the separation of double-stranded RNA during replication and transcription. By unwinding RNA, it ensures that the RNA-dependent RNA polymerase (RdRp) can access the template strand for effective replication and transcription of the viral genome.

Answer 58

Nucleoside Triphosphatase (NTPase): NTPase activity is critical for hydrolysing nucleoside triphosphates (NTPs) to provide the energy required for various RNA processing events, including unwinding RNA during replication and facilitating the binding of RdRp to RNA templates. This activity supports the overall efficiency of viral RNA synthesis.

Answer 59

RNA Exonuclease: nsp14 functions as an exonuclease, providing proofreading capabilities by removing mismatched nucleotides from the newly synthesized RNA. This activity is crucial for maintaining the fidelity of RNA replication, reducing the mutation rate, and enhancing the stability of the viral genome.

Answer 60

Replication complexes are specialized structures within infected cells that act as sites for viral RNA synthesis, often referred to as "RNA factories." Composed of viral and cellular proteins, these complexes are associated with modified cytoplasmic membranes. Viral proteins, particularly non-structural proteins nsps 3, 4, and 6, modify existing membranes and stimulate the production of new membranes, facilitating the formation of replication complexes. Nucleocapsid protein is also abundant at these sites, enabling the encapsidation of newly synthesized genomic RNA. Overall, replication complexes provide an organized environment crucial for viral RNA synthesis and processing.

Answer 61

When a coronavirus infects a host cell, it manipulates the host's cellular machinery, including the ER, to create these membrane-bound vesicles. These vesicles form a network within the host cell and help create an environment conducive to viral RNA synthesis and protein production. The RVN provides a scaffold for the formation of double-membrane vesicles (DMVs), which are critical for viral replication. The viral replicase complex, which is responsible for synthesising the viral genome, assembles on these membranes. After replication, the viral proteins and newly synthesized RNA are packaged into new viral particles, which are then transported to the cell surface in vesicles derived from the RVN.

Answer 62

Replicative intermediate: RNA molecule on which one or several growing RNA strands are being synthesized * The growing strand typically forms base-pairing to template RNA only near their growing 3’ end.

Answer 63

Discontinuous transcription in coronaviruses is the process by which subgenomic (sg) mRNAs are produced from subgenomic negative strand mRNA templates. Key steps include: RNA polymerase initiates transcription at the 3' end of the genomic RNA and synthesises negative strand templates until it encounters a transcription-regulating sequence (TRS), which signals transcription termination. In murine hepatitis virus, short TRS sequences are found at the 5' ends of genes 2-7 and at the 3' end of the leader sequence. Upon reaching a TRS, the polymerase pauses, dissociates the nascent RNA chain, and jumps to a TRS at the end of the leader sequence, forming RNA-RNA hybrids. The polymerase can pause at any TRS, allowing for the production of multiple sg mRNAs from different genomic regions. Each subgenomic negative strand serves as a template to generate a corresponding positive strand mRNA, facilitating the expression of viral proteins essential for replication and pathogenesis.

Answer 64

The M (membrane) and E (envelope) proteins are essential for the formation of the coronavirus envelope during budding: Virus-Like Particle Formation: M and E proteins alone can generate enveloped virus-like particles in the ERGIC, indicating their crucial role in viral assembly. Viral RNA Packaging: The C-terminal tail of M interacts with packaging signals in the nucleocapsid protein (N), ensuring that only full-length viral RNA is included in virions. Incorporation of Other Proteins: The spike (S) and hemagglutinin-esterase (HE) proteins are incorporated into the membrane through interactions with M, contributing to the complete viral structure. Glycosylation: Envelope proteins are glycosylated as they pass through the Golgi apparatus, which is important for their stability. Virion Release: Mature virions are packaged into vesicles and targeted to the plasma membrane for release, completing the viral life cycle.

Answer 65

The viral genome consists of a linear positive-sense single-stranded RNA (ssRNA) that ranges from 7 to 8.9 kilobases in length. This genome features a single open reading frame (ORF) that encodes a large polyprotein, which is subsequently cleaved into distinct functional proteins by viral proteases. This organization allows for efficient use of the viral genome and a streamlined replication process.

Answer 66

Picornaviruses possess a naked icosahedral capsid, which lacks a lipid envelope, making them more resilient in the environment. With a diameter of approximately 30 nanometers, their capsid comprises 60 copies of three to four structural proteins: VP1, VP2, VP3, and VP4. These proteins form a compact structure, where VP1, VP2, and VP3 create the external shell, while VP4 is located internally, contributing to the integrity and stability of the virion.

Answer 67

Entry Mechanism: Picornaviruses enter host cells by binding to specific cellular receptors through canyons or loop regions on their surface. This interaction facilitates receptor-mediated endocytosis or direct membrane penetration, allowing the viral RNA to be released into the cytoplasm. Translation Strategy: The 5' non-coding region of the viral RNA contains an internal ribosome entry site (IRES), which enables translation initiation independent of the typical 5' cap structure found in cellular RNAs. The IRES recruits ribosomal subunits and various cellular proteins to initiate translation at an internal site, effectively hijacking the host's translational machinery.

Answer 68

Following translation, viral RNA replication occurs within multiprotein complexes that associate with cellular vesicles. These vesicles proliferate rapidly upon infection, serving as sites for viral replication. A full-length negative-strand RNA, known as the antigenome, is synthesized and then used as a template for generating new positive-strand RNA genomes. This replication process is initiated by VPg, a protein covalently linked to the RNA, which acts as a primer for RNA synthesis.

Answer 69

Assembly and Release: During assembly, the precursor protein VP0 is cleaved into VP2 and VP4, contributing to the formation of the mature virion. Assembly pathways can vary, including threading the RNA genome into the procapsid or associating pentamers with the RNA to form a provirion. Mature virions are released from the host cell through lysis, allowing them to spread and infect additional cells.

Answer 70

Flaviviruses are enveloped particles approximately 50 nanometers in diameter, displaying a smooth, “golf ball” appearance due to their icosahedral symmetry. The viral capsid measures 25-30 nanometers and contains a spherical nucleocapsid. The envelope is comprised of 180 copies of two glycoproteins, M and E, which form heterodimers and contribute to the virus's structural integrity without protruding from the surface.

Answer 71

The genome of flaviviruses consists of a linear positive-sense single-stranded RNA (ssRNA) approximately 10-11 kilobases long. It is capped at the 5’ end but lacks a poly(A) tail at the 3’ end. The RNA is translated as a single polyprotein, which undergoes proteolytic cleavage to yield one capsid protein (C), two envelope proteins (M and E), and seven non-structural proteins. This genomic organization is similar to that of picornaviruses but distinct from togaviruses.

Answer 72

Flavivirus entry begins with the E protein, which mediates receptor binding and membrane fusion. While no specific cellular receptor has been definitively identified, viruses can enter host cells via endocytosis in clathrin-coated vesicles. Additionally, antibody-dependent enhancement (ADE) may occur, allowing virus-antibody complexes to enter cells expressing immunoglobulin Fc receptors, leading to more severe disease manifestations, such as dengue haemorrhagic fever.

Answer 73

Once inside the cytosol, the viral RNA is translated into a polyprotein, which is cleaved to produce functional proteins. The capsid protein precursor (anchC) is directed to the endoplasmic reticulum (ER), where it is processed. The precursor membrane protein (prM) associates with the E protein to form a heterodimer that protects E from premature activation. Following this, non-structural proteins facilitate the establishment of RNA replicase complexes, which synthesize a complementary negative-strand RNA to serve as a template for the production of additional positive-strand RNA molecules for translation, replication, and packaging.

Answer 74

Flavivirus assembly occurs at intracellular membranes, primarily the ER-Golgi interface. Once the immature virion reaches the Golgi, exocytosis begins and the prM protein is cleaved by the host enzyme furin. This cleavage converts prM into M, producing the mature viral particle capable of infecting host cells. This maturation process is critical, as the pr peptide is only released upon exposure to neutral pH outside the cell, ensuring proper viral functionality

Answer 75

Togaviruses are spherical enveloped particles approximately 70 nm in diameter, exhibiting a fringe of projections or spikes. Their nucleocapsid and envelope glycoproteins are organized in icosahedral symmetry, comprising 240 heterodimers of glycoproteins E1 and E2 in the envelope and 240 copies of the capsid protein.

Answer 76

The viral genome consists of a linear ‘+’ sense ssRNA of about 9.7 to 11.8 kb, featuring a 5’ methylated cap and a 3’ poly(A) tail of roughly 70 nucleotides. The genome encodes four non-structural proteins responsible for viral RNA synthesis, translated directly from the genomic RNA as a polyprotein, which is then processed by host and viral proteases. Additionally, five structural proteins are produced from a subgenomic mRNA, including one capsid protein, three envelope proteins, and a small hydrophobic protein.

Answer 77

The E glycoprotein facilitates the virus's entry into host cells through receptor-mediated endocytosis, interacting with receptors like laminin and heparan sulfate, which are components of the extracellular matrix. Following attachment and entry into clathrin-coated vesicles, a drop in pH within the endosome triggers conformational changes in the E1/E2 heterodimer, leading to the fusion of the viral envelope with the endosomal membrane and the release of the nucleocapsid into the cytoplasm.

Answer 78

Once in the cytoplasm, the viral RNA genome is released for translation. For instance, in Sindbis virus, the non-structural proteins are synthesized as a P123 polyprotein, and occasionally, a readthrough of the stop codon produces P1234. These proteins are critical for synthesizing full-length antigenome RNA, with early synthesis catalyzed by P123 and nsP4 (RNA-dependent RNA polymerase). As replication proceeds, the partially cleaved non-structural proteins transition to producing plus-strand RNA.

Answer 79

The structural proteins undergo post-translational modifications, including cleavage by host signal peptidases and furin proteases, and are palmitoylated within the Golgi apparatus. The capsid proteins interact with the cytoplasmic tails of the envelope proteins located in the plasma membrane, facilitating viral assembly. Finally, togaviruses exit the host cell by budding, completing their infectious cycle.

Answer 80

Lipid Envelope with Glycoproteins: The viral envelope, derived from the host cell membrane, contains glycoprotein (GP) spikes that enable cell attachment and fusion. Nucleocapsid: Inside the envelope, the nucleocapsid is a helical structure made of RNA and nucleoproteins, which protect and stabilize the viral genome. Negative-Sense RNA Genome: The virus has a single-stranded, non-segmented negative-sense RNA genome (~19 kb), encoding seven proteins essential for infection, including glycoprotein (GP), nucleoprotein (NP), matrix proteins (VP24 and VP40), and polymerase (L). Matrix Proteins: VP40 and VP24 stabilize the virus, with VP40 aiding assembly and budding, while VP24 also helps modulate host immune responses.

Answer 81

The first recorded Marburg virus outbreak occurred in Germany in 1967, where laboratory workers handling monkey tissues fell ill; 32 cases resulted in a 22% mortality rate. Ebola virus (Ebolavirus, EBOV) was first identified in 1976 with outbreaks in Zaire (now the Democratic Republic of Congo) and Sudan. Since then, Ebola and Marburg viruses have re-emerged sporadically, mainly in Africa, causing severe, often fatal diseases with mortality rates up to 90%.

Answer 82

- Zaire ebolavirus (ZEBOV): The most deadly, linked to the largest outbreak in 2014-2015 in West Africa, which resulted in 11,000 deaths and 28,000 cases. - Sudan ebolavirus (SEBOV), - Cote d'Ivoire ebolavirus (CIEBOV), - Bundibugyo ebolavirus (BEBOV), - Reston ebolavirus (REBOV) (the latter is non-pathogenic in humans).

Answer 83

- Transmission occurs through direct contact with bodily fluids (blood, urine, feces, vomit, and semen) of infected persons, making close contacts like family members and healthcare workers the most at risk. - Symptoms include severe haemorrhagic fever, liver dysfunction, intravascular coagulation, cytokine release, and vascular leakage, often leading to shock. - Human-to-human aerosol transmission is low, and outbreaks often self-limit due to short transmission periods.

Answer 84

A highly effective rVSV-ZEBOV vaccine is available for Zaire ebolavirus, with global stockpiles ready for rapid deployment. No effective vaccine exists for SEBOV.

Answer 85

Filoviruses have compact, linear genomes with seven genes arranged in a conserved order, each coding for a single viral protein except for the glycoprotein gene (GP), which can be cleaved into different forms. These genomes include conserved sequences that regulate transcription termination, polyadenylation, and reinitiation to ensure the virus produces separate transcripts for each protein. Most viral proteins are incorporated into the virion structure.

Answer 86

1. Nucleocapsid Protein (NP): Encapsidates the RNA genome, forming the core structure. 2. RNA Polymerase Cofactor (VP35): Essential for viral RNA synthesis and immune evasion. 3. Matrix Protein (VP40): Mediates virus assembly and budding from the host cell membrane. 4. Envelope Glycoproteins (GP): GP can be cleaved into GP1 and GP2 for receptor binding and membrane fusion or secreted as sGP (which may play a role in immune evasion). 5. Minor Nucleocapsid Protein (VP30): Functions as a transcription factor. 6. Membrane Protein (VP24): Helps regulate immune responses and may aid in assembly. 7. RNA Polymerase (L): Catalyses viral RNA synthesis.

Answer 87

1. Transcription Initiation: Viral RNA polymerase starts at the genome’s 3’ end. 2. mRNA Transcription: Initially, when nucleocapsid protein (NP) levels are low, polymerase frequently terminates after transcribing short segments, releasing a leader RNA. It then scans to find the next gene's mRNA start site for re-initiation (an inefficient process). 3. mRNA Processing: Each mRNA transcript receives a 5' methylated cap and a polyA tail for stability, similar to cellular mRNAs. 4. Genome Replication Initiation: Once NP levels are sufficient, the polymerase switches to genome replication: * It synthesises a full-length antigenome (positive-strand) RNA. * This antigenome acts as a template for producing new negative-strand RNA genomes. * During this phase, polymerase ignores termination signals, producing continuous full-length RNA. 5. Genome Encapsulation: New genomes are encapsidated with NP to form stable nucleocapsids, ensuring efficient genome replication only when enough NP is present.

Answer 88

Inclusion bodies in the context of filovirus replication are cytoplasmic structures where viral replication and assembly are concentrated. These structures contain viral RNA, proteins, and nucleocapsids, creating an organized space for replication and protecting viral components from cellular degradation. They play a crucial role in efficiently producing and packaging new viral particles before they exit the cell

Answer 89

Filoviruses, like the Ebola virus, use RNA editing to produce different glycoproteins from a single gene, adding flexibility to their replication strategy. The viral RNA polymerase “stutters” over a sequence of seven uracils (U), sometimes inserting an additional adenine (A), which shifts the reading frame. This polyadenylates the previous subgenome, and promotes the continued synthesis of the next. This editing process yields two main products: sGP (secreted glycoprotein), the more common form, lacking the transmembrane domain, and GP (glycoprotein), the full-length version involved in viral entry. Occasionally, additional variations like ssGP (smaller sGP) can also arise from differing numbers of inserted A residues.

Answer 90

Furin, a cellular protease, cleaves (activates) GP into GP1 and GP2. GP1 binds to the host cell receptor, while GP2 facilitates the fusion of the viral envelope with the host cell membrane.

Answer 91

Asialoglycoprotein Folate receptor-α Integrins DC-SIGN

Answer 92

pseudotypes Such pseudoviruses are widely used to study the interactions between viral envelope proteins and host cell receptors under controlled and safe conditions. By eliminating the risk of uncontrolled viral replication, pseudotypes allow researchers to investigate entry mechanisms, neutralising antibody responses, and host-cell tropism without the biohazard risks associated with fully infectious viruses.

Answer 93

sGP is released from infected cells and is found in serum of infected patients → can be used as a biomarker and as a potential vaccine / antiviral target. * Function is not entirely clear: * Considered non-structural but may substitute as a structural protein by forming a complex with GP2 * Some pseudotyping data shows that it may limit GP cytotoxicity → more efficient replication and infectivity (?, speculation) * Acts as a soluble factor that targets elements of the host defence system e.g. binding to antibodies and contribute to immunosuppression

Answer 94

The secreted glycoprotein (sGP) in filoviruses, particularly Ebola virus, likely subverts the immune system by acting as a decoy. Because it is secreted in large amounts, sGP can bind to and neutralise antibodies that would otherwise target the membrane-bound GP on the virus surface, effectively diverting immune responses away from the infectious virion. This binding can help mask the virus from neutralising antibodies, allowing it to evade immune recognition. Additionally, sGP can interfere with signalling and interaction among immune cells, further impairing the host's immune response and contributing to the virus's immune-evasive strategies.

Answer 95

The minor nucleocapsid protein VP30 activates viral mRNA synthesis by reversing an inhibitory stem-loop structure at the start of the NP gene, which otherwise blocks RNA polymerase from initiating transcription. Experimental disruption of this stem-loop eliminates the dependency on VP30, though the precise mechanism of action remains unclear. VP30 is present in nucleocapsids, suggesting it may interact directly with the transcription machinery or RNA structures to relieve the inhibition.

Answer 96

VP40 is the most abundant viral protein, located on the cytoplasmic side of the plasma membrane or the inner side of the viral envelope. Similar to matrix proteins in other enveloped viruses, VP40 bridges the envelope glycoproteins to nucleocapsids. Expressing VP40 alone in mammalian cells can produce virus-like particles that bud from the plasma membrane, and this process involves interactions with cellular proteins like Nedd4 (ubiquitin ligase) and Tsg101 (a component of the complex directing vesicles to endosomes). These interactions help direct VP40 to sites of viral assembly at the plasma membrane, facilitating virion formation and release.

Answer 97

It is showing an ebolavirus: - Uses NPC1 as receptor, common in Ebolavirus

Answer 98

Recrudescence: recurrence of disease / symptoms after a period of inactivity

Answer 99

Ebolavirus (EBOV) recrudescence involves periodic spikes in antibodies months after clinical recovery, suggesting latent infections that can lead to disease recurrence. Vaccinating survivors may enhance protective antibody responses and help prevent these relapses. Persistent EBOV in survivors, despite blood clearance, can result in sexual transmission and severe inflammatory conditions in immune-privileged areas like the eyes and brain. Lethal relapses have occurred in some survivors, highlighting the need for ongoing monitoring to prevent further outbreaks.

Answer 100

Influenza viruses belong to the family Orthomyxoviridae, derived from the Greek words ortho (correct or normal) and myxa (mucus), reflecting their ability to attach to mucoproteins on host cell surfaces. These enveloped viruses can appear either quasi-spherical or filamentous, with a diameter ranging from 80 to 120 nm. The envelope is acquired from the host membrane during the budding process. Inside, influenza viruses feature compact helical nucleocapsids, which house the viral genetic material.

Answer 101

Public Health Measures: Many of the measures implemented to control the spread of COVID-19, such as mask-wearing, social distancing, and enhanced hygiene practices, also effectively reduced the transmission of influenza. School and Workplace Closures: The closure of schools and remote work arrangements limited social interactions, which are critical for the spread of both viruses. Increased Vaccination: There was a heightened awareness of respiratory illnesses during the pandemic, leading to increased flu vaccination rates in some populations. Travel Restrictions: International travel restrictions and reduced movement between regions likely curtailed the spread of flu viruses. Viral Competition: The predominance of SARS-CoV-2 during the pandemic may have limited the opportunities for influenza viruses to circulate and establish infections

Answer 102

- Uni-12 and Uni-13 are highly conserved sequences (universal primers) that are self-complementary - Genome segments 1-6 each encode for a single protein: three are RNA polymerase subunits (PA, PB1, PB2), envelope glycoprotein HA, neuraminidase (NA). * In some influenza viruses, a 2nd reading frame on segment 2 codes for a shorter protein PB1- F2 (localizes to mitochondria and enhances apoptosis). - Alternate splicing can occur in mRNA transcribed from segments 7 and 8 → different proteins * Segment 7: = Matrix protein M1 = Envelope protein M2 * Segment 8: = Non-structural proteins NS1 and NS2

Answer 103

Function: - HA binds to sialic acid residues on cell surface receptors (mucoproteins) of various cell types. Mediates viral fusion with the endosome after endocytosis. Key Properties: - Agglutination of RBCs: HA can agglutinate red blood cells (RBCs) because they express sialic acid. This is used in the hemagglutination assay. Type I transmembrane protein: HA spans the viral membrane with a single hydrophobic domain. Trimer formation: HA forms trimers on the virus surface that are essential for the fusion process. Fusion Mechanism: - Upon endocytosis, the acidic environment of the endosome triggers a conformational change in HA trimers. This change enables fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the host cell. Importance: - HA is crucial for both viral attachment and entry into host cells. It is a key target for antiviral strategies.

Answer 104

Membrane Fusion of Influenza Viruses - Influenza virus entry into host cells is a complex process involving the hemagglutinin (HA) protein on the viral surface. The N-terminal of HA is exposed to the outside of the virion, while a hydrophobic transmembrane domain near the C-terminal anchors it in the viral envelope. Fusion of the viral envelope with the host cell membrane occurs in two key steps: 1. Activation by Proteolytic Cleavage: - The HA protein must first be cleaved by cellular proteases (like furin or trypsin) into two subunits: * HA1: The surface subunit that binds to sialic acid on the host cell, facilitating viral attachment. * HA2: Anchored in the viral membrane, it contains a hydrophobic fusion peptide crucial for the fusion process. 2. Conformational Change After Acidification: - After the virus is endocytosed into the host cell, the endosome's acidic environment triggers HA to facilitate the fusion of the viral envelope with the endosomal membrane which allows the release of the viral genome into the cytoplasm.further changes that enable fusion: - The M2 protein forms an ion channel in the viral membrane, allowing protons (H⁺) to enter the virus. This acidification weakens the interaction between the matrix protein (M1) and the nucleocapsids, leading to the release of the viral genome. M2 is a small (~97 amino acids) tetrameric protein that creates a small pore in the viral envelope. Its structure includes: * External N-terminal domain * Transmembrane domain * Larger internal base domain - The tryptophan (W41) residue in M2 acts as a "gate," interacting with aspartate (D44) to keep it closed. When a histidine (H37) residue in the protein is protonated, it triggers a conformational change, opening the gate and allowing H⁺ conductance. Antiviral Target: - The M2 ion channel is a key target for antiviral drugs, such as Amantadine, which block the proton entry. However, resistance to these drugs can arise due to mutations in the M2 protein.

Answer 105

Influenza Virus RNA Replication and Transcription The replication of influenza virus RNA occurs in the nucleus, where the viral nucleocapsids (comprised of the RNA genome and nucleoproteins (NPs)) enter and facilitate mRNA synthesis and RNA replication. Key Steps in RNA Replication and mRNA Synthesis: 1. Nucleocapsid Entry into the Nucleus: - The nucleocapsid, which contains the RNA genome and the trimeric RNA polymerase complex (PA, PB1, and PB2), carries nuclear localization signals that interact with importin-α for nuclear entry via the nuclear pore complex. 2. Viral mRNA Synthesis: - The RNA polymerase complex transcribes the viral RNA in the nucleus. PB1 binds conserved sequences at the 5' and 3' ends of the viral RNA. The PB2 subunit recognizes capped cellular pre-mRNAs, and PB1 cleaves the pre-mRNA at the 5' cap, using the fragment as a primer for viral mRNA synthesis. This cap-stealing mechanism allows the virus to use the host cell’s mRNA capping machinery to create viral mRNAs. 3. Transcription of 8 Viral mRNAs: - Transcription generates eight viral mRNAs: * Segments 1-6 are directly exported to the cytoplasm. * Segments 7 and 8 undergo alternative splicing in the nucleus, using splicing consensus sequences recognized by the host's splicing machinery. Approximately 90% of these mRNAs are unspliced, encoding the M1 and NS1 proteins, which are abundant. The spliced forms encode M2 and NS2, which are less abundant. 4. RNA Replication: - Genome replication involves creating a + strand (antigenome), which is complexed with NP. This antigenome is used to produce full-length copies of the viral genome. This process does not involve the “stuttering” mechanism seen in mRNA synthesis and remains poorly understood. The antigenome RNA is then copied back into the genome RNA. 5. NP Balance and Transcription vs. Replication: - The amount of NP in the nucleus regulates the balance between transcription and replication. As NP binds to growing RNA chains, the NP concentration decreases, prompting more mRNA synthesis to produce additional NP, which is then imported back into the nucleus. NP binds to uncapped genome RNA but not to the capped mRNA, which helps distinguish the two processes. 6. Nucleocapsid Export and Packaging: - After replication, nucleocapsids are exported from the nucleus in a complex with the matrix protein (M1) and NS2. Newly synthesized M1 is imported into the nucleus, where it forms a complex with the nucleocapsids. NS2 binds to M1 and contains a nuclear export signal, which is recognized by exportins to export the complex to the cytoplasm. 7. Cytoplasmic Events: - In the cytoplasm, the newly synthesized viral proteins are translated, and the viral genome is packaged into new virions, completing the viral replication cycle.

Answer 106

Influenza Virus Assembly and Release Influenza virus assembly and release involves a well-coordinated process that ensures the formation of new virions and their subsequent release from the host cell. Viral Envelope Protein Trafficking: - HA, NA, and M2 are synthesized in the endoplasmic reticulum (ER) and Golgi apparatus, where they are processed and trafficked to the plasma membrane. These proteins accumulate in specialized regions of the membrane known as lipid rafts, which are membrane microdomains enriched in cholesterol and sphingolipids. Interaction with M1 and Nucleocapsids: The cytoplasmic tails of HA, NA, and M2 proteins interact with the M1 matrix protein that is associated with the nucleocapsids. This interaction is crucial for assembling the viral particle by bringing together the viral RNA genome and the structural components required for virion formation. Genome Packaging: The virus packages one copy of each genome segment into the nucleocapsid, ensuring that the resulting virions contain a complete set of viral RNA for the next round of infection. Viral proteins bind to specific RNA sequences within the nucleocapsid, guiding the RNA segments into bundles that are then encapsulated in the viral particle. Budding and Envelope Acquisition: As the virus begins to bud from the host cell, it acquires its envelope from the plasma membrane. During this process, the neuraminidase (NA) protein plays a key role by cleaving sialic acid from the cellular receptors that are bound to the viral HA, preventing the newly formed virions from being tethered to the host cell. This enzymatic activity allows the virus to escape the host cell surface and be released into the extracellular space. Role of M2 in Budding: M2, a small ion channel protein, is involved in the final steps of the budding process. Evidence suggests that M2 helps in the "pinching off" of the budding virion from the plasma membrane, effectively releasing the new virions from the host cell. Neuraminidase (NA) Structure: NA is a type II transmembrane protein, meaning it has a short cytoplasmic N-terminal, a membrane-spanning domain, and a long C-terminal domain extending outward from the viral envelope. This orientation is the reverse of HA, which is a type I transmembrane protein. The NA's enzymatic activity cleaves sialic acid residues, facilitating viral release by preventing re-attachment of virions to the host cell.

Answer 107

Antigenic Changes in Influenza Viruses: - Influenza A viruses are classified into subtypes based on two surface proteins: hemagglutinin (HA) and neuraminidase (NA). There are 18 HA subtypes (HA1 → HA18) and 11 NA subtypes (NA1 → NA11). Most of these subtypes infect birds, with only a few, notably H1N1 and H3N2, circulating in humans. Influenza A (H1N1 and H3N2) and Influenza B (which has two strains) are included in seasonal flu vaccines. Types of Antigenic Change: - Antigenic change in the virus can lead to the emergence of new strains and the potential for pandemics. There are two primary mechanisms of antigenic change: 1. Antigenic Drift: 2. Antigenic Shift: Notable Strains: - H5N1 (known as bird flu or avian influenza) is a rare, highly pathogenic strain (HPAI) that can infect humans, primarily through contact with infected birds. It has a 60% mortality rate in humans, though it is not easily transmitted from person to person.

Answer 108

This is a slow and continuous accumulation of point mutations in the HA and NA genes. These mutations gradually alter the viral surface proteins, allowing the virus to evade host immunity and leading to the emergence of new strains that can cause seasonal outbreaks. Antigenic drift is why flu vaccines need to be updated every year to match the circulating strains.

Answer 109

- This occurs when two or more different influenza subtypes infect the same host and exchange genetic material. This reassortment of genes can create a completely new viral subtype, with novel HA and/or NA proteins that may be drastically different from previously circulating strains. Because the human population has little to no immunity to these new subtypes, antigenic shift can potentially lead to pandemics. - A significant example of antigenic shift occurred in 2009, when a new H1N1 strain emerged as a result of genetic reassortment. This new strain spread globally, causing approximately 20,000 deaths and leading to an urgent vaccine development campaign.

Answer 110

1. Attachment and Entry _Hemagglutinin (HA),_ a glycoprotein on the virus surface, binds to _sialic acid_ residues on the surface of the host cell. This binding occurs mainly on respiratory epithelial cells in the case of human influenza. Once bound, the virus is internalized through _endocytosis,_ entering the cell inside a vesicle. The low pH inside the endosome triggers a _conformational change in HA, facilitating the fusion of the viral envelope_ with the endosomal membrane, allowing the viral genome to be released into the host cell's cytoplasm. 2. Uncoating and Release of Viral Genome After fusion, the viral genome, packaged in nucleocapsids, is released into the host's cytoplasm. The _M2 ion channel protein_ in the viral envelope allows protons to enter the virus, leading to acidification inside the virion. This acidification causes the dissociation of the M1 matrix protein, releasing the RNA segments (genome) into the cytoplasm. 3. Nuclear Entry The viral nucleocapsids (RNA wrapped in nucleoproteins) are _transported to the nucleus._ This transport is facilitated by nuclear _localization signals on the NP and RNA polymerase_ proteins, which interact with the host's _importin-α,_ enabling entry through the nuclear pore complex. 4. Transcription and Replication of Viral RNA Inside the nucleus, the viral RNA polymerase _(composed of PB1, PB2, and PA)_ transcribes the viral genome into mRNA. The mRNA is used for protein synthesis, while the genomic RNA serves as a template for replication to produce new viral genomes. This process involves the use of host pre-mRNAs for _cap-stealing to initiate transcription, a unique feature of the influenza virus._ 5. Translation of Viral Proteins The mRNAs are exported from the nucleus to the cytoplasm, where the host's ribosomes translate them into viral proteins. These proteins include the structural components _(like HA, NA, M1, M2)_ and RNA polymerase subunits, which are necessary for the assembly of new viral particles. 6. Viral Assembly In the cytoplasm, newly synthesized viral proteins and genomic RNA are assembled into new nucleocapsids. HA, NA, and M2 viral proteins are trafficked through the Golgi apparatus and incorporated into the plasma membrane in lipid rafts, while the M1 matrix protein binds to the nucleocapsids to form the viral core structure. 7. Budding and Release The new viral particles bud off from the host cell membrane, acquiring an envelope that contains the viral HA, NA, and M2 proteins. Neuraminidase (NA) cleaves sialic acid residues on the host cell surface, preventing newly formed virions from reattaching to the host cell and allowing for their release. The host cell is often damaged or destroyed during this process, and the newly released virions can go on to infect new cells.

Answer 111

- The genome contains repeated (R) sequences at both the 5' and 3' ends (150-200 nt). - Between the R sequences are the U5 (untranslated 5') and U3 (untranslated 3') regions, which vary in size. - A specific cellular tRNA binds to the genomic RNA at the primer binding site (PBS) near the U5 region, which is crucial for reverse transcription. - The genome also contains the Ψ (packaging) sequence, essential for RNA packaging into virions. - 5'SS and 3'SS represent splice sites involved in splicing the RNA during transcription

Answer 112

Retroviruses were reclassified into two subfamilies and eleven genera

Answer 113

Retroviruses have three main groups of proteins derived from the gag, pol, and env genes: 1. Gag (Group-Specific Antigen): - MA (Matrix): Associates with the viral envelope. - CA (Capsid): Forms the protein shell surrounding the RNA genome (often referred to as p24). - NC (Nucleocapsid): Protects the RNA genome and assists with reverse transcription. 2. Pol (Polymerase): - PR (Protease): Cleaves viral polyproteins into mature functional proteins. - RT (Reverse Transcriptase): Converts the RNA genome into DNA. - IN (Integrase): Facilitates the integration of the viral DNA into the host genome. 3. Env (Envelope): - SU (Surface Unit): Also called gp120, it binds to host cell receptors. - TM (Transmembrane Unit): Also called gp41, it mediates fusion of the viral envelope with the host cell membrane. - These viral proteins are initially synthesized as polyproteins, which are cleaved into mature functional proteins by protease during or after the assembly of new virions.

Answer 114

polypurine tract

Answer 115

Retroviruses enter host cells through the fusion pathway, with the virus's envelope protein (SU) interacting with specific cellular receptors, determining the virus's tropism. For example, HIV-1 uses CD4 and the chemokine receptors CCR5 and CXCR4 as receptors to enter host cells. Once the virus binds to the cell surface, the envelope either fuses with the plasma membrane directly or undergoes endocytosis, followed by pH-dependent fusion within the endosome. This fusion releases the viral core into the cytoplasm, allowing the next steps of the viral lifecycle to begin. During entry, a conformational shift in the SU protein exposes the hydrophobic N-terminal of the TM protein (transmembrane), which is then inserted into the host cell membrane, facilitating fusion.

Answer 116

Once inside the cytoplasm, the retrovirus begins the process of reverse transcription. This is the conversion of its RNA genome into DNA, catalyzed by the viral enzyme reverse transcriptase (RT). RT is a dimeric enzyme with two key enzymatic activities: * RNA-dependent DNA polymerase: Converts the RNA into DNA. * Ribonuclease H (RNase H): Degrades the RNA strand of the RNA hybrid formed during reverse transcription. This process begins with a cellular tRNA acting as a primer, enabling the reverse transcriptase to synthesize the complementary DNA strand. The DNA copy of the viral genome is then ready for integration into the host genome.

Answer 117

Retrovirus integration is a crucial step where the viral proviral DNA is incorporated into the host cell's genome. This process is facilitated by the viral enzyme integrase (IN), which is packaged inside the preintegration complex in the virion. Upon release into the host cell, integrase binds to the two ends of the proviral DNA and catalyzes the integration process by bringing the ends of the viral DNA close to the host genome. Key Steps in Integration: - Cleavage of the Viral DNA: Integrase removes two nucleotides from the 3' end of each strand of the viral DNA. This leaves free 3' hydroxyl (-OH) groups. The removal of these 3' nucleotides is crucial because the 3' OH is needed for the next step of DNA ligation. - Cleavage-Ligation Reaction: Integrase facilitates a cleavage-ligation reaction, bringing the 3’ OH ends of the viral DNA in close proximity to the phosphodiester linkages of the host DNA. The viral and host DNA strands are joined through this reaction, with the viral DNA attaching to the host DNA at random sites. - Gaps Left Behind: The joining process creates small gaps: * A 4-6 nucleotide single-stranded gap in the host DNA. * A 2 nucleotide unpaired gap in the viral DNA. - Host Repair Mechanism: Host DNA repair enzymes then fill in the gaps: They synthesize the missing nucleotides in the host DNA. The two unpaired nucleotides on the viral DNA are removed, generating a direct repeat of host DNA at the integration site.

Answer 118

In the late phase of retrovirus replication, the viral RNA and protein synthesis occurs, leading to the assembly of new virions. Proviral DNA can remain in a latent, unexpressed state for extended periods, requiring activation by specific transcription factors to initiate gene expression. Key Points in Retrovirus Transcription: 1. Transcription Regulation: The U3 region of the proviral DNA contains specific sequences that interact with cellular transcription machinery, helping to control when transcription occurs. This allows the virus to respond to the host cell's conditions. 2. LTR (Long Terminal Repeat) Elements**: * The TATA box at the U3/R junction directs cellular RNA polymerase II to initiate transcription of the viral genome. * The Poly(A) signal at the R/U5 boundary directs the cleavage of the RNA transcript, and poly(A) is added by host cell enzymes, creating a polyadenylated mRNA. 3. RNA Transcription: The transcription of proviral DNA produces a full-length RNA transcript identical to the viral genome. This serves as both the template for viral protein production and as the genomic RNA for new virions.

Answer 119

1. Unspliced mRNA: This full-length RNA encodes the Gag and Gag/Pol polyproteins, which are essential for viral assembly and replication. 2. Spliced mRNA: This shorter RNA is processed to produce Env proteins, which are essential for the virus’s ability to infect new cells.

Answer 120

Splicing of the viral RNA allows for the generation of different protein products from the same viral genome, with unspliced RNA encoding structural proteins like Gag and Pol, while spliced RNA is responsible for producing the envelope proteins needed for virus entry into host cells. In summary, retrovirus transcription is regulated by sequences in the LTR that interact with the host machinery, producing full-length RNA that is used both for protein synthesis and as the viral genome. Splicing of the viral RNA generates different mRNAs for producing essential proteins, enabling the virus to complete its lifecycle.

Answer 121

1. Receptor Binding: CD4 serves as the primary receptor for HIV-1 and is found on T lymphocytes and monocytes/macrophages. In addition to CD4, a co-receptor such as CCR5 (for macrophage-tropic viruses) or CXCR4 (for T cell-tropic viruses) is required for viral entry. The co-receptor usage is determined by variations in the viral SU protein (gp120), which dictates which receptor the virus can interact with. Ligands that block CD4 or co-receptors can prevent viral entry by interfering with this binding process. 2. Conformational Changes and Fusion: Binding of gp120 to CD4 triggers a conformational change in the viral envelope protein. This exposes additional regions of gp120 that bind to the co-receptor, further promoting fusion. The fusion domain (gp41) is then exposed, which facilitates the insertion of the viral membrane into the host cell membrane. Fusion occurs, bringing the viral and host cell membranes into close proximity and releasing the viral core into the host cell's cytoplasm. 3. Nucleocapsid Uncoating: Once inside the host cell, the nucleocapsid partially disassembles, allowing access to the cellular nucleotide pool, essential for viral replication. 4. Reverse Transcription: The viral reverse transcriptase (RT) replicates the HIV RNA genome, converting it into DNA. The reverse transcription process generates small mutations because RT lacks a proofreading mechanism, leading to genetic variability. These mutations can help the virus evade the immune system or drugs, complicating treatment. 5. Transport to the Nucleus: Unlike other retroviruses, HIV-1 actively directs its proviral DNA into the nucleus for integration into the host genome. This can occur even in non-dividing cells, ensuring a productive infection. Proteins such as MA (matrix), Vpr, and IN (integrase) form the preintegration complex: MA contains a nuclear import signal and interacts with importins to facilitate nuclear entry. Vpr and IN directly interact with the nuclear pore to mediate transport into the nucleus. 6. Integration: Once in the nucleus, the integrase (IN) enzyme catalyzes the integration of the proviral DNA into the host genome, allowing the virus to replicate along with the host cell's DNA.

Answer 122

Integration into Host DNA: - After HIV reverse transcribes its RNA genome into DNA, the viral proviral DNA is integrated into the host cell's genome. This integration can lead to either an active or latent infection. - In the latent state, the virus remains dormant within the host cell, with the viral genome present but not actively producing viral proteins. This allows the virus to evade detection by the immune system, as infected cells do not express viral proteins that could trigger an immune response. Regulation of Latency: - Transcriptional control elements in the HIV-1 LTR (long terminal repeat) region regulate whether the virus remains latent or becomes active. - The U3 region of the LTR contains binding sites for cellular transcription factors like NFκB (nuclear factor kappa B) and NFAT (nuclear factor of activated T-cells). These transcription factors control whether the virus is transcribed into RNA. - The activity of these transcription factors is influenced by cellular stimuli, such as T cell receptor (TCR) engagement. When the TCR is activated (for example, during immune responses), transcription factors like NFκB are activated and translocate to the nucleus, which can trigger the transition from latent to active viral replication. Challenges in Cure: Because the virus can hide in latent reservoirs within infected cells, where it does not actively replicate or produce viral proteins, the immune system often fails to recognize and eliminate these infected cells. As a result, patients with HIV-1 must remain on antiretroviral therapy (ART) for life, as the virus can reactivate at any time if the conditions are right (such as through T cell activation), leading to continued replication and disease progression.

Answer 123

- Tat (Transactivator of Transcription): Tat is a small, basic protein (86 amino acids) produced from doubly-spliced HIV-1 mRNA, enhancing viral genome transcription. It binds to the Tat-responsive element (TAR) on nascent RNA, not proviral DNA, influencing RNA polymerase activity. In Tat's absence, RNA polymerase II lacks processivity. When Tat binds TAR, it recruits Cdk9 and Cyclin T, phosphorylating RNA polymerase II and boosting transcription efficiency. - Cis-Acting Repressive Sequences (CRS): CRS in the gag, pol, and env regions inhibit RNA transport from the nucleus. These sequences must be overcome for effective gene expression. - Rev and mRNA Transport: After proviral integration, doubly-spliced viral RNA produces early proteins like Tat, Rev, and Nef, essential for transcription and mRNA export. Rev binds to the Rev Response Element (RRE) on unspliced or single-spliced mRNA, facilitating their transport from the nucleus despite CRS, enabling translation of structural proteins. Doubly-spliced mRNA, encoding early proteins, does not contain CRS and is transported without Rev. - Shuttling of Rev: Rev shuttles between the nucleus and cytoplasm, using nuclear import and export signals to transport unspliced or single-spliced mRNAs for translation into structural proteins.

Answer 124

Function: Vif increases the infectivity of HIV virions by counteracting a host cell antiviral factor called APOBEC3G, a cytidine deaminase that can mutate viral DNA, reducing viral infectivity. Mechanism: APOBEC3G is incorporated into virions and deaminates cytosine (C) to uracil (U) in viral DNA, causing mutations that affect viral proteins. Vif induces ubiquitination and degradation of APOBEC3G by the proteasome, thereby preventing this antiviral action and increasing the infectivity of the virus.

Answer 125

Function: Vpr enhances HIV replication by facilitating the entry of the preintegration complex (PIC) into the host nucleus and enhancing replication. Mechanisms: Vpr interacts with the Gag protein (C-terminal), aiding in the packaging of cellular uracil DNA glycosylase (UNG), which removes uracil from DNA. Vpr can also arrest cells at the G2 stage of the cell cycle, likely by promoting the degradation of cellular proteins required for progression to the M phase. This arrest benefits HIV replication since transcription is most active during the G2 phase.

Answer 126

Function: Vpu enhances the release of progeny virions from infected cells by facilitating the degradation of CD4 and counteracting host antiviral factors that tether the virus to the host cell. Mechanisms: - CD4 retention: CD4 in the cytoplasm binds to the viral gp160 protein (precursor to gp120), preventing its incorporation into new virions. Vpu binds β-TrCP, triggering CD4 degradation through the proteasome, allowing for the release of gp160 and increasing the surface expression of gp41 and gp120. - Antiviral counteraction: Vpu also combats tetherin, a host protein that tethers virions to the cell surface and promotes their degradation. By degrading tetherin, Vpu enhances virion release, allowing HIV to efficiently bud off from the plasma membrane. In the absence of Vpu, virions accumulate on the surface of the cell (partial budding), hindering release.

Answer 127

Nah vaccinations eradicated it but the CDC and VECTOR still have viable smallpox samples

Answer 128

Assembly of Immature Virions (IV): The assembly of vaccinia virions begins with de novo synthesis of rigid, crescent-shaped membrane structures, which are not connected to pre-existing internal membranes. These crescents are destined to become the mature viral envelope. As the crescents mature, they transition into spherical shapes (immature virions, IV), encapsulating the viroplasm, an electron-dense material that contains viral core proteins. 1. Formation of Viral DNA Core: - The viral DNA enters the spheres, forming discrete, dense cores (also known as nucleoids). - As the immature virions continue to develop, they undergo rearrangement of materials inside the spheres to create internal core structures and lateral bodies, ultimately acquiring their final shape and becoming mature virions (MVs). 2. Intracellular Maturation: - The majority of newly synthesized virions remain in the cell as intracellular MVs. These are not released but can play a role in cell-to-cell spread through direct cell contact. 3. Extracellular Virion (EV) Formation: - A small proportion of the MVs undergo further maturation to become extracellular virions (EVs), which are exported and used for cell-to-cell spread. - These MVs are enveloped by Golgi-derived cisternae, which add additional lipid layers and viral proteins to the virions, preparing them for export. 4. Transport and Release via Actin Tails: - Wrapped viruses (WVs), which have two bilayer membranes (one from the Golgi and one from the viral envelope), are transported to the plasma membrane. - This transport is facilitated by actin tails, which are induced by viral proteins that promote the polymerization and depolymerization of actin. These actin tails help propel the viral particles towards the cell surface. - When the outermost membrane of the wrapped virus fuses with the host cell membrane, the extracellular virus (EV) is released. In some cases, the EV remains tethered to the cell surface via microvilli projections, assisting in further spreading the virus. 5. Lipids and Envelope Proteins: - Lipids required for the viral envelope are delivered to the crescent structures from the endoplasmic reticulum (ER) in the form of small vesicles (micelles), which contain the necessary viral envelope proteins. This ensures the viral envelope is properly assembled and functional.

Answer 129

The actin-based propulsion mechanism enhances viral dissemination by physically delivering virions to nearby cells, reducing their exposure to neutralising antibodies or other extracellular antiviral factors. This ensures more efficient cell-to-cell spread of the virus.

Answer 130

Inactivated viruses are viruses that have been rendered non-infectious through specific treatment methods, allowing them to be used safely in applications like vaccine development. The process involves cultivating the virus in controlled conditions, purifying it to remove contaminants, and then inactivating it using chemical agents such as formaldehyde or organic solvents, or by exposing it to high temperatures. This method preserves the virus’s structural integrity and antigenic properties, enabling it to elicit an immune response without causing disease. Examples include: Hep A vaccine, Rabies vaccine

Answer 131

Subunit vaccines consist of purified viral proteins that are immunogenic but lack any viral genome, ensuring they cannot cause infection. These vaccines are relatively easy and cost-effective to produce using recombinant technologies like genetic cloning, which allows for the large-scale synthesis of specific antigenic components. Examples: 1. Hepatitis B Vaccine 2. Human Papillomavirus (HPV) Vaccine 3. Pertussis Vaccine

Answer 132

VLPs mimic the structure of a virus but lack genetic material, ensuring they cannot replicate in host cells. They present antigens in a virus-like configuration, making them more effective at eliciting immune responses than simple subunit proteins. Like subunit vaccines, they often require adjuvants and boosters due to their lack of replication. Examples: - Gardasil (HPV Vaccine) - Cervarix (HPV Vaccine) - Hepatitis E Vaccine

Answer 133

1. mRNA Vaccines: Deliver synthetic mRNA encoding the spike protein; highly immunogenic with rapid production but require ultra-cold storage (e.g., Pfizer, Moderna). 2. Viral Vector Vaccines: Use adenoviruses to deliver spike protein genes; stable under refrigeration but may face reduced efficacy due to pre-existing immunity (e.g., AstraZeneca, Johnson & Johnson). 3. Protein Subunit Vaccines: Contain purified viral proteins with adjuvants; safe and established but less immunogenic, often requiring boosters (e.g., Novavax). 4. Inactivated Vaccines: Use chemically or heat-inactivated virus; traditional, stable, and safe but less potent immune responses (e.g., Sinovac, Covaxin). 5. DNA Vaccines: Use plasmid DNA encoding spike protein; easy to produce and store but still emerging with limited long-term data (e.g., ZyCoV-D).

Answer 134

A dead-end host

Answer 135

Inhibition of DNA Polymerase: - Acyclovir triphosphate competes with deoxyguanosine triphosphate (dGTP) as a substrate for viral DNA polymerase. It preferentially binds to the viral DNA polymerase due to its higher affinity compared to the host DNA polymerase. Chain Termination: - Once incorporated into the viral DNA, acyclovir lacks the necessary 3'-hydroxyl group for further elongation. This leads to premature termination of viral DNA synthesis, halting replication. Viral Specificity: - The reliance on viral thymidine kinase for activation ensures that acyclovir is selectively activated in infected cells. Healthy cells, lacking viral TK, phosphorylate acyclovir inefficiently, reducing its toxicity to uninfected tissues.

Answer 136

Ritonavir: a successful protease inhibitor of HIV-1 → viral protease has an unusual substrate specificity that is not found in human proteases - _Peptidomimetic_ drug → mimics a peptide and iterative modifications to drug improved its pharmacokinetics (uptake, distribution, degradation, clearance) * Designed based on natural substrate of HIV protease → a region in the Gag-Pol polyprotein

Answer 137

This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature non-infectious viral particles. Protease inhibitors are almost always used in combination with at least two other anti-HIV drugs

Answer 138

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