Virology Final Flashcards
Virion:
A complete virus particle outside of the host cell
What does a virion consist of? (3)
A nucleic acid genome
A protective protein coat (capsid)
Some contain a lipid envelope
Nucleic acid genome
Encodes for proteins that allow the virus to replicate in the host and transmit from one cell to another or one host to another.
Once inside of a host cell a virus will undergo uncoating of its _____ to release its genetic material inside the cell. This goes on to the hijacking of host machinery in order to synthesise viral ____ and ____. After the virus has replicated its genetic material (DNA or RNA), the viral structural proteins surround and ____ the newly made genome to protect it. Once the viral genome is encapsulated, the fully formed virus particles are considered “_____ viruses,” ready to infect other cells
capsid
mRNA
proteins
enclose
new progeny
Viral structural proteins ______ the newly replicated genome → new progeny virus particles
encapsidate
How does mutation connect with evolutionary lineage?
Use a constant mutation rate and cross reference it with the molecular clock
In DNA what are both the positive and negative strands responsible for?
“+” strand virus encodes for protein, the “-” strand encodes for the complementary strand
What are potential issues of having RNA genomes
- mRNA must be synthesised from a RNA template instead of DNA template
- RNA genome must be replicated
* This is problematic because the host cell doesn’t have the machinery to do that therefore most RNA viruses encode their own RNA-dependent RNA polymerases
_______ have RNA genome which gets converted to DNA by the host cell using ____ ____ (cDNA copy of RNA) encoded by viral genome e.g. HIV
Retroviruses
reverse transcriptase
Describe the hershey chase experiment
demonstrated that DNA is the carrier of genetic information. They used two scenarios: one with radioactively labeled DNA (using phosphorus-32) and another with labeled proteins (using sulfur-35). When bacteriophages infected cells, only the radioactive DNA entered the cells, while the labeled proteins remained outside. This indicated that DNA, not protein, carries genetic material, confirming its role in heredity.
Describe Tobacco Mosaic Virus
Tobacco Mosaic Virus (TMV) is the first identified plant virus, responsible for tobacco mosaic disease. Dimitri Ivanovski (1892) and Martinus Beijerinck (1898) discovered that the agent causing the disease could pass through fine filters that blocked bacteria, revealing the existence of infectious agents smaller than bacteria. TMV has a rod-shaped structure consisting of RNA surrounded by a protein coat and leads to mottled discoloration in infected plants. This research laid the foundation for virology and techniques like filter sterilization, which are still used in labs today. TMV’s discovery also influenced the identification of other viruses, such as those causing foot-and-mouth disease and yellow fever.
Describe plaque assays
- Measure concentration of bacteriophages by their ability to lyse bacteria.
- Bacterial growth is measured using a spectrophotometer; intact bacteria refract light, making cultures appear cloudy.
- Phages bind to bacterial cells, replicate, and release progeny, leading to cell lysis and loss of light diffraction (clearing).
- In solid cultures, phages infect surrounding cells, causing repeated lysis and forming clear areas (plaques) against uninfected cells.
- Plaques are counted as plaque-forming units (PFU), indicating the number of infectious virus particles in a suspension.
How are plaque assays done in eukaryotic cells
How are red blood cells commonly used to measure/detect viruses?
How do you interpret hemagglutination assays?
What is the implication of electron microscopy in viral analysis
Virus particles can be seen and counted by electron microscopy (cannot tell infectious vs not)
E.g. virus particles are mixed with an electron dense stain (e.g. phosphotungstate, uranyl acetate) → viruses do not take up the stain → observed as light image against a dark background (negative staining)
Describe negative staining
In negative staining, the stain does not bind to the virus itself. Instead, the negatively charged stain surrounds the virus particles, creating a dark background. The virus particles appear lighter in contrast, which makes them easier to visualise under an electron microscope. The stain does not bind to positively charged molecules on the virus surface.
How do plaque assays and electron microscopy relate to the ratio of infectious particles (if desired)
Describe the multiplicity of infection (MOI)
Multiplicity of infection (MOI) = Number of infectious virus particles per susceptible cell e.g MOI of 10-100 PFUs / cell is often used
Infect with excess virus to ensure that each cell receives at least one infectious particle.
Infects nearly all cells simultaneously, leaving few uninfected cells
Limitation: steps in replication cycle overlap → difficult to study
How do you calculate the percentage of the viral genome’s coding capacity used by the protein?
- Determine the nucleotide requirement for the protein:
* Each amino acid is encoded by 3 nucleotides (a codon).
* The protein is 100 amino acids long.
* So, the number of nucleotides required to encode the protein is: 100 amino acids×3=300 nucleotides - Convert the genome size to nucleotides:
* The genome size is given in kilobases (kb), which means 1 kb = 1000 nucleotides.
* The viral genome is 6.3 kb, so it contains: 6.3 kb×1000=6300 nucleotides - Calculate the percentage of the genome used by this protein:
* (300 nucleotides / 6300 nucleotides)×100=4.76%
Look at this
Analysis of viral macromolecules (proteins, mRNAs, genomes) can reveal the detailed
pathways of virus replication. Can be studied using assays involving what?
- Radiotracers (radioactive compounds incorporated into viral or cellular DNA, RNA,
proteins, lipids, carbohydrates) - Antibodies against specific proteins
- Molecular hybridization (labelled DNA or RNA probes)
- PCR
- Gel electrophoresis
- Microscopy
Describe the viral replication cycle
Describe the binding to cell receptor phase of the viral replication cycle
Describe viral entry in the replication cycle?
Describe early viral gene expression and replication in the replication cycle
Describe late gene expression and assembly of virions in the replication cycle
Describe the exit phase of viral replication cycle
Describe Baltimore classifications of viruses
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.
____: a rigid symmetrical container for viral genome –> generally made with viral proteins
____: capsid with enclosed viral genome
____: Lipid bilayer membrane surrounding capsids / nucleocapsids
Capsid
Nucleocapsid
Envelope
Describe genetic economy
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.
What shapes / symmetries do viruses express
Cubic
Icosahedral
Describe parvovirus
- 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.
More complex capsids have repeating subunits interacting in a ___-___ (nearly
equivalent) manner → symmetrical distribution of protein subunits on the surface of a capsid
such that they form similar interactions (not identical)
quasi-equivalent
Know this
Protein subunits can move slightly to accommodate distortions therefore they have built in flexibility
Describe capsids with helical symmetry
Describe the calculations associated with helical capsids
Describe viral envelopes
- 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.
In ____ if you took a virus particle coming from viral plasma membrane, buds from the cell surface would have the same cholesterol and phospholipid concentrations in proportion to that of the plasma membrane from which it originates.
influenza
Describe Flaviviruses
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
Describe the ERGIC compartment
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
Describe viral glycoproteins
- Most viruses will have Viral glycoproteins (proteins with a sugar attached) embedded in the lipid bilayer. Can be any sort of shape
Describe the characteristics of envelope viral glycoproteins
- Large glycosylated (the controlled enzymatic modification of an organic molecule, especially a protein, by addition of a sugar molecule) external domain (ectodomain)
- Hydrophobic transmembrane anchor domain
- 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
Describe influenza virus in the context of this class
- 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
Describe type I integral membrane proteins
- 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).
Describe type II integral membrane proteins
- 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.
Describe the signal sequence on the envelope protein which directs membrane insertion
- 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
Describe budding as a mechanism of viral envelope acquisition
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
Describe packaging of viral genomes
- Genome packaged within capsid
- In some viruses, capsids assemble around the genome, and in some the genome is inserted into a preformed shell
Describe scaffolding proteins
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
What is a procapsid?
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.
A viral genome _____ is a long continuous DNA or RNA molecule composed of repeated genome units linked end to end. It forms during viral replication and is typically processed into individual genomes before packaging into new viral particles.
concatemer
______ are protein-coated structures that enclose and protect a virus’s genetic material, either DNA or RNA. They play a crucial role in viral stability and assist in the delivery of the viral genome into host cells during infection.
Nucleocapsids
What are the three types of capsids?
What is the function of packaging signals?
Describe core proteins
Describe matrix proteins in the context of viral budding
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.
Describe how budding can be driven by envelope glycoproteins
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.
Describe virion disassembly
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.
Virus classification is based on what?
- 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
Virus hosts can be divided into what 6 categories of organisms
- Bacteria
- Archaea
- Lower eukaryotes (fungi, protozoa, algae)
- Plants
- Invertebrates
- Vertebrates (including humans)
Describe virus species
Know these generalisations
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.
Describe certain traits that are notable about plant viruses
Describe unique mechanisms of transcription where capsids act as tiny intracellular machines
Describe viruses with reverse transcriptase step in their replication cycle
- 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.
Describe viruses with dsRNA genomes
Describe satellite viruses, nucleic acids and viroids
Look at this
Describe evolutionary origin of viruses
- 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
There are limited number of viral genes that encode for what?
- Structural proteins
- Proteins that stimulate DNA replication enzymes in
host - Proteins that recognize and bind to viral DNA and
assemble cellular replication machinery
What are the seven major virus groups
- 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)
Describe viral entry
Describe viral entry again
Describe entry of enveloped viruses - fusion
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
Describe entry of non-enveloped viruses
Describe cell-cell transmission
Describe virological synapses
Describe syncytia
Describe intracellular channels such as plasmodesmata
Describe virus entry attachment phase
- 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)
How do host cell receptors interact with surface components of a virion
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.
Affinity of individual interactions may be fairly low but attachment can be exceptionally tight.
Can you think of a reason why?
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.
Describe receptor-mediated endocytosis
Describe Non-enveloped virus entry via lysis
Describe Macropinocytosis
- 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)
Describe acidification as a function of viral entry
Describe membrane fusion
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
Describe membrane fusion
Describe class I fusion proteins
Describe Class II fusion proteins
Describe class III fusion proteins
Look at this
Describe avoidance of target cell membrane lysis by alternatively using pore formation
Describe intracellular transport
Describe the import of viral genome into the nucleus
What are other methods of viral genomes into the nucleus
Describe the entry and uncoating of adenovirus
Describe the virus replication cycle and how host cells have antiviral strategies
Describe some methods of preventing virus
What are some ways of inhibiting uncoating of capsids
Describe positive-strand RNA Viruses
Describe coronaviruses
Describe the coronavirus genome
Describe the purpose of N protein in regards to coronavirus nucleocapsids
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.
Describe the S1 subunit of coronavirus spike proteins
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.
Describe the S2 subunit of coronavirus spike proteins
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.
Describe the types of cellular receptors that alphacoronavirus spike proteins bind to
-
Describe the types of cellular receptors that betacoronavirus spike proteins bind to
Describe the basics of coronavirus entry
- 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
What are the functions of TMPRSS2 (tempress 2; on cell surface)
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
Describe the replicase gene (Gene 1)
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.
Describe the synthesis of coronavirus viral proteins
A ribosomal frameshift allows ORF1b to be translated, producing pp1ab, which is then cleaved by viral proteases to yield several non-structural proteins (nsps). These nsps form the replication-transcription complex (RTC), which directs viral RNA synthesis and includes enzymes like RNA-dependent RNA polymerase (RdRp), helicase, and exonuclease, essential for accurate genome replication and subgenomic RNA transcription.
Explain the function of pp1a
pp1a yields 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.
Describe the function of pp1ab
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.
Describe papain-like cysteine proteinases
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.
Describe the function of RNA-dependent RNA polymerase (ORF1b, nsp12)
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
Describe the function of RNA helicase
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.
Describe the function of nucleoside triphosphatase
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.
Describe the function of RNA exonuclease (ORF1b, nsp14)
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.
Describe replication complexes
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.
Describe reticulovesicular network
Coronavirus infection induces significant rearrangement of cytoplasmic membrane architecture, forming a network known as the reticulovesicular network.
This network includes double-membrane vesicles (DMVs), convoluted membranes (CMs), and double-membrane spherules (DMSs), which are observable by electron microscopy
Describe coronavirus genome replication
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.
Describe coronavirus subgenomic mRNA
Describe discontinuous transcription in coronaviruses
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.
Describe polymerase transcription abortion
Describe Coronaviruses Assembly and Release of Virions
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.
Describe the genomic characteristics of picornaviruses
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.
Describe the structure of picornaviruses
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.
Describe the entry and translation of picornaviruses
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.
Describe the replication of picornaviruses
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.
Describe the assembly and release of picornaviruses
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.
Just read this
Describe the morphological characteristics of flaviviruses
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.
Describe the genomic structure of flaviviruses
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.
Describe the entry and attachment of flaviviruses
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.
Describe the replication process of flaviviruses
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.
Describe the assembly and release of flaviviruses
Flavivirus assembly occurs at intracellular membranes, primarily the ER-Golgi interface. Newly synthesized viral particles are processed as they transit through the Golgi apparatus and are released from the host cell by exocytosis. Cleavage of the prM protein by cellular furin protease occurs just before virion release, converting immature particles into mature virions. This maturation process is critical, as the pr peptide is only released upon exposure to neutral pH outside the cell, ensuring proper viral functionality
Describe the structure of togaviruses
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.
Describe the genomic composition of togaviruses
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.
Describe the entry mechanisms of togaviruses
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.
Describe the replication of togaviruses
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.
Describe the assembly and exit of togaviruses
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.
Describe the genomes and proteins of filoviruses
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.
Describe the Key Filovirus Proteins:
- Nucleocapsid Protein (NP): Encapsidates the RNA genome, forming the core structure.
- RNA Polymerase Cofactor (VP35): Essential for viral RNA synthesis and immune evasion.
- Matrix Protein (VP40): Mediates virus assembly and budding from the host cell membrane.
- 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).
- Minor Nucleocapsid Protein (VP30): Functions as a transcription factor.
- Membrane Protein (VP24): Helps regulate immune responses and may aid in assembly.
- RNA Polymerase (L): Catalyses viral RNA synthesis.
Describe the Key Steps in Filovirus Replication
- Transcription Initiation: Viral RNA polymerase starts at the genome’s 3’ end.
- 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).
- mRNA Processing: Each mRNA transcript receives a 5’ methylated cap and a polyA tail for stability, similar to cellular mRNAs.
- 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. - Genome Encapsulation: New genomes are encapsidated with NP to form stable nucleocapsids, ensuring efficient genome replication only when enough NP is present.
Describe inclusion bodies
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
Describe filovirus RNA editing
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 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.
describe filovirus attachment and entry
Filovirus attachment and entry are mediated by the glycoprotein (GP), which is processed through the endoplasmic reticulum (ER) and Golgi, where it undergoes glycosylation. Once at the plasma membrane, the GP precursor is cleaved by cellular furin protease into GP1 (ectodomain) and GP2 (transmembrane) subunits, held together by a disulfide bridge. The cleaved GP assembles into trimers on the virion surface, enabling attachment to a range of cellular receptors (e.g., asialoglycoprotein, folate receptor-α, integrins, and DC-SIGN) for binding and entry. Macropinocytosis then transports the virus into the cell, where GP2’s fusion peptide facilitates fusion with vesicle membranes, potentially triggered by low pH
Describe the function of sGP
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
It is showing an ebolavirus:
- Ebolavirus uses the VP35 protein as an antiviral sensor and replicates in viral factories within the host cell, providing a unique strategy for evading the immune response.
- Ebolavirus has been shown to utilize cellular pathways such as actin-dependent transport, while Marburgvirus does not primarily rely on this mechanism for viral release.
Describe the structure of influenza viruses
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.
What are the types of influenza viruses
Whats the Host range of Influenza A viruses. (not overtly important)
Describe the disease and spread of influenza viruses
Describe the genome of influenza viruses
- 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
Describe the membrane fusion of influenza viruses
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:
- 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. - Conformational Change After Acidification:
- After the virus is endocytosed into the host cell, the endosome’s acidic environment triggers 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.
Describe influenza virus RNA replication
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:
- 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. - 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. - 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. - 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. - 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. - 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. - 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.
Describe influenza virus assembly and release
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.
Describe antigenic changes in influenza viruses
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:
- Antigenic Drift:
- 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.
Describe the trans-species viral transmission of influenza throughout the years
Describe vaccines for influenza viruses
Describe the seven steps of the influenza virus life cycle
- 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. - 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. - 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. - 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. - 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. - 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. - 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.
Describe the structure of retroviruses
- 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
Describe the three proteins of retroviruses
Retroviruses have three main groups of proteins derived from the gag, pol, and env genes:
- 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. - 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. - 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.
Describe the early development of retroviruses
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.
Describe the origin of AIDS
How is HIV transmitted
Describe HIV
Describe the structure of HIV
What are the mechanisms of HIV
Describe the acute infection and clinical latency of HIV
What are the three different types of progressors if HIV is left untreated
What are the first two steps in retrovirus reverse transcription
What are steps 3-4 in retrovirus reverse transcription
What are steps 5-9 in retrovirus reverse transcription
Describe retrovirus integration
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.
Describe retrovirus transcription
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:
- 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.
- 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. - 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.
Describe retrovirus splicing
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.
Just look at this
What are the structural and non-structural proteins of HIV
Describe the splicing of HIV primary transcripts
Describe HIV entry and replication
- 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. - 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. - Nucleocapsid Uncoating:
Once inside the host cell, the nucleocapsid partially disassembles, allowing access to the cellular nucleotide pool, essential for viral replication. - 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. - 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. - 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.
Describe HIV transcription
Tat (Transactivator of Transcription):
- Tat is a small, highly basic protein (86 amino acids) produced from doubly-spliced HIV-1 mRNA. It plays a critical role in enhancing the transcription of the viral genome.
- Tat is localized in the nucleus and binds to the Tat-responsive element (TAR) located at the transcription start site of HIV RNA. It binds to nascent RNA, not the proviral DNA, which was one of the first discoveries showing that newly synthesized RNA can influence the activity of RNA polymerase.
- In the absence of Tat, RNA polymerase II lacks processivity, meaning it cannot effectively elongate the RNA. However, when Tat binds to TAR, it recruits cellular Cdk9 and Cyclin T, which phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, enhancing its processivity and transcription efficiency. This process significantly boosts the transcription of the HIV genome.
Cis-Acting Repressive Sequences (CRS):
- CRS sequences are present in the gag, pol, and env regions of the viral genome and act to inhibit RNA transport from the nucleus to the cytoplasm. These sequences must be overcome for efficient gene expression.
Rev and mRNA Transport:
- Once proviral DNA is integrated, the viral RNA produced is initially doubly-spliced (about 2 kb), which is essential for the synthesis of early proteins like Tat, Rev, and Nef. These proteins are necessary for both viral transcription and the export of mRNAs encoding structural proteins.
- The Rev protein is crucial for the cytoplasmic transport of viral mRNAs that code for structural proteins. Rev binds to the Rev Response Element (RRE) present on unspliced or single-spliced viral mRNAs, which are otherwise restricted from transport due to the presence of CRS. Rev facilitates the transport of these mRNAs out of the nucleus.
- Doubly-spliced mRNA, which encodes early proteins, does not have CRS and can be transported normally without the need for Rev.
Shuttling of Rev:
- Rev is a dynamic protein that shuttles continuously between the nucleus and cytoplasm. It contains both nuclear import and export signals, allowing it to mediate the transport of unspliced or single-spliced mRNAs out of the nucleus for translation into structural proteins.
Describe Vif (Viral Infectivity Factor):
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.
Describe Vpr (Virion Protein R):
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.
Describe Vpu (Viral Protein Unique to HIV-1):
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.
Describe HIV budding and maturation
Just continuously review and integrate this HIV life cycle thing
Describe the general structure of poxviruses
What are the two subfamilies of poxviruses
Describe the disease and history of poxviruses
Describe the disease progression of poxviruses
Describe variolation
Who pioneered vaccination of poxviruses
Describe the structure of poxvirus virions
Describe the differences in morphology between the two virion forms of poxviruses
Describe the structure of mature poxviruses (think glycosaminoglycans)
Describe the structure of extracellular poxviruses
Just look at this poxvirus shit
Describe the three transcription factors of poxviruses
Describe the early genes of poxviruses (first 3 steps of poxvirus replication)
What do you call poxvirus DNA replication factories
Describe the first 5 steps of concatemer formation of poxviruses
Describe the last three steps of concatemer formation of poxviruses
How do poxvirus DNA concatemers get resolved into monomers
Describe postreplicative genes
Describe poxvirus assembly and exit
What is actin used by vaccinia viruses
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.
- 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). - 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. - 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. - 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. - 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.
Look at this lifecycle of poxviruses
