Lecture 3+4 Virus infection Flashcards
(45 cards)
1
Q
Baltimore classification of viruses
A
Groups viruses according to the differences in morphology, genetics and how the mRNA is produced during the replicative cycle of the virus. mRNA is by definition a positive strand.
2
Q
Group I viruses
A
dsDNA
- +/- DNA –> + mRNA
Example: herpes simplex/adenovirus
3
Q
Group II viruses
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(+) ssDNA
- + DNA –> +/- DNA –> + mRNA
Example: parvovirus
4
Q
Group III viruses
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dsRNA
- +/- RNA –> + mRNA
Examples: reovirus/rotavirus
5
Q
Group IV viruses
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(+) ssRNA.
- + RNA –> - RNA –> + mRNA
Example: poliovirus
6
Q
Group V viruses
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(-) ssRNA
- - RNA –> + mRNA
These viruses carry RNA-dependent RNA-polymerase in the virion so that they can make mRNAs upon infecting the cell. They may be:
- Segmented e.g. Influenza
- Non-segmented e.g. Rhabdoviruses (rabies)
Example: influenza/rabies/ebola
7
Q
Group VI viruses
A
(+) ssRNA (RT)
- + RNA –> - DNA –> +/- DNA –> + mRNA
It must be converted, using the enzyme reverse transcriptase, to dsDNA.
Example: HIV/retrovirus
8
Q
Group VII viruses
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(+/-) dsDNA (RT)
- partial +/- DNA –> +/- DNA –> mRNA
They act as mRNA but are also converted back into dsDNA genomes by reverse transcriptase.
Example: hepatitis B virus
9
Q
Virions
A
Virus particles
10
Q
Positive-strand RNA virus
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Isolated RNA is directly infectious.
RNA is directly translatable by ribosomes into functional proteins: virus particles have a positive RNA which is sensed as mRNA. It can directly be translated.
- Do not need to package the viral RNA polymerase in the virus particle to be infectious
- Can be used as mRNA to make the viral RdRP
- Make a copy of negative polarity (replication intermediate) which is then used as template to make many new genomic RNA molecules
Example: polio
11
Q
Negative-strand RNA virus
A
Isolated RNA is not infectious.
RNA is not directly translatable: first copy it from – to + RNA
- Because the antisense (or non-coding) strand is encapsidated in the virions
- Carry RNA-dependent RNA polymerase (RdRP) that promotes the synthesis of the coding strand when the virus has entered the cell and has released its negative-stranded viral genome in the cytoplasm
- RdRP is encoded in viral genome, more made in infected cell to allow efficient viral replication
Example: flu
12
Q
Poliovirus
A
Positive stranded, non-segmented RNA virus.
Primarily infection in the GI tract. It can affect the nervous system.
13
Q
Influenza A virus
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Negative stranded segmented RNA virus
14
Q
HIV
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Retrovirus: copying its RNA genome into integrated DNA prior to protein expression
15
Q
Linear RNA
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Polar molecule with a 5’ and a 3’ end.
- Ribosomes scan (+) RNA molecules from the 5’ to the 3’ end and translate the open reading frame into a protein in the same direction.
- All RNA molecules are also synthesized from the 5’ to the 3’ end, and for that they need another RNA (or DNA) molecule as template.
- From the template a reverse complement is being made, so this template is copied from its 3’ to 5’ end.
- (+) sense RNA molecules can serve as template for the synthesis of (-) sense RNA copies (and vice versa).
- The plus and minus strands are called reverse complements of each other.
16
Q
How are viral proteins being made
A
Bacteria can make several proteins from one mRNA, whereas eukaryotic ribosomes normally only translate the first ORF, which is the one closest to the 5’ end of the mRNA. This means that eukaryotic organisms in general cannot translate polycistronic mRNAs (mRNAs with more than one open reading frame (ORF). RNA viruses have developed several strategies to circumvent this problem:
- They may have a single ORF encoding a large polyprotein, which is later cleaved in functional proteins (e.g. poliovirus).
- Some RNA viruses have segmented genomes, where every RNA molecule carries the information for one protein (e.g. influenza virus).
- To make sub-genomic mRNAs (using a full length (-) RNA molecule as template) to express all genes (e.g. Chikungunya virus).
- Making use of differential splicing (e.g. HIV) resulting in individual mRNA molecules for the various proteins
Combinations of these strategies may also occur.
17
Q
Gene functions of the poliovirus
A
-
ssRNA (+) molecule, of approx. 10,000 nt
–> non-segmented
–> strategy IV - 5’-terminal protein and a 3’-polyA tail
- Contains one long coding region
–> Open reading frame: ORF - Is translated into large ‘polyprotein’, which is later cleaved into 10 individual, smaller, functional, viral proteins
–> Is cut into individual subunits - T=3: three different coat proteins that together form the surface of the particle
–> VP1, VP2, VP3
–> VP4 is located under the surface layer
18
Q
Structural proteins
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Proteins of the viral capsid
19
Q
Non-structural proteins
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Not being part of the virus particle, but only formed and function inside the infected cell. They are needed for virus replication.
20
Q
Genome replication of poliovirus
A
Replication of the polioviral RNA, (making new genome copies), occurs in membrane vesicles in the cytoplasm induced by this virus.
- Binding of the poliovirus coat protein VP1 to host poliovirus cell receptors (PVR) on the cell surface (transmembrane protein) mediates endocytosis
- Due to binding, capsid undergoes a conformational change (disassembly) in the poliovirus coat to open a pore in the host endosomal membrane: viral RNA release and enters the cytoplasm
-
VPg is removed from the viral RNA, followed by translation by ribosomes of the positive-stranded RNA genome to make a polyprotein
–> VPg is a small protein that binds viral RNA and is necessary for synthesis of viral positive and negative strand RNA, attached at the 5’ end - Replication via dsRNA occurs in viral factories made of membrane vesicles derived from the ER (copy)
- The dsRNA genome is transcribed/replicated to produce mRNA and new ssRNA(+) genomes
- Polyprotein is cleaved into the smaller, mature viral proteins by 2A and 3B
- New viral RNA is packaged into preassembled procapsids (capsid formation)
- Cell lysis (bursts open) and millions of virus particles release
- During replication, first a negative copy is being made using the genomic (+) strand as a template. Temporarily, this results in a double-stranded replication form.
- The new (-) strand then serves as template to make more (+) strands.
- These new (+) strands can either be translated or become packaged in progeny virus particles.
21
Q
What are the final products of translation of the poliovirus
A
- Viral RNA-dependent RNA polymerase (RdRP) –> Responsible for the synthesis of (a few) (-) strand RNA molecules that serve as replication intermediates to make many plus strand RNA’s, most of which are encapsulated using the newly produced coat proteins to form progeny virus particles.
-
Coat proteins VP1-VP4.
–> The poliovirus viral protein VPg is covalently attached to the 5’ end of the genomic RNA (red dot in figure), of both the (+) and (-) strand copies.
–> VPg stands for “Viral protein genome-linked”).
–> This protein is removed before translation of the (+) strand RNA. This virus has a poly(A) tail at the 3’ end.
Like the genomic RNA of all picornaviruses, the polioviral RNA contains a single, long open reading frame (ORF), which is translated into a single “polyprotein”.
- The functional, smaller proteins are generated by autocatalytic, proteolytic cleavages of the polyprotein by the 2A and 3C proteases (which are themselves part of the polyprotein).
22
Q
Which three intermediate cleavage products are generated in the translation of the poliovirus
A
- P1: delivers the coat proteins (VP1 – VP4) (structual proteins)
- P2: 2A protease cleaves polyprotein and eIF4G
–> eIF4G is required for initiation of translation by allowing ribosomes to the 5’ end of host mRNAs
–> Translation of host mRNA is inhibited → translational machinery comes available for translation of poliovirus RNA
–> The process by which an invading virus blocks an essential cellular mechanism that prevents cellular protein synthesis is called “host shut-off”. - P3: VPg (3B) and the viral RdRP (protein 3D) are derived from P3.
6 non-structural proteins: not being part of the virus particle
VPg: Viral Protein genome-bound
23
Q
Influenza virus A
A
Major example of a negative stranded segmented RNA virus
- Has a lipid ‘envelope’
–> All particles are slightly different; are not icosahedral
–> Membrane is derived from its host cell - 3 “serotypes”: A, B and C;
–> Subtypes (H1N1) - Serotype A most important: causing worldwide epidemics (pandemics)
- Coughing is needed for the virus to transmit
24
Q
The schematic structure of influenza A virus
A
- HA recognizes neuraminic acid = sialic acid
-
8 segments of viral RNA that make up the genome
–> All segments are different and essential for virus replication
–> Each genome segment is covered with nucleoprotein (NP) forming 8 nucleocapsids inside viral envelope
–> The segments carry a copy of the viral RdRp-complex to allow independent transcription (required because of negative polarity)
–> Needs RNA polymerase to kick off replication -
2 Envelope glycoproteins with opposite functions. Determine the antigenic properties of the virus
–> HA: to get in (entry)
–> NA: to get out (egress) - Matrix protein (M1 & M2): stabilize the envelope
Eight different RNA molecules packaged together in a virus particle form the viral genome and these all encode other viral proteins. The 3 largest RNA segments (RNA1-3) encode the 3 subunits of the viral RdRP (these subunits are called PA, PB1 and PB2). Other RNA segments encode a number of non-structural proteins.
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Indicate types of influenza A virus
- H: 16 serotypes
- N: 9 serotypes
- H1N1
- H3N2
- H5N1
- H7N7
The segmentation is important for synthesis of all viral proteins and a crucial property that allows influenza viruses to genetically *adapt* quickly through **recombination**.
26
Replication cycle of Infuenza virus A
Needs to fuse its own membrane with the membrane of the host cell. Influenza virus is packaged in a viral envelope that fuses with the plasma membrane. This way, the virus can exit the host cell without killing it.
--> Replication RNA in the **nucleus**
1. HA protein binds to a cell surface receptor and is invaginated into the cell at **clathrin-coated pits** and taken up in an endosome. The viral **M protein** is then released making a pore in the endosomal membrane allowing the viral ribonucleoprotein-complexes to enter the cytoplasm, from where they are transported to the nucleus.
2. in the **nucleus** the viral RNAs are transcribed into **(+) strand copies** by the co-packaged **RdRP**. The (+) strand copies can be **translated**, allowing the synthesis of structural and non-structural proteins.
A few (+) strand copies will also be replicated and new (-) genome fragments will be generated that will be packaged in RNP's
3. The viral mRNAs for the **HA** and **NA** envelope **glycoproteins** are translated at the rough **ER**, where also **glycan** (sugar) side chains are added. The glycan side chains are further modified, and the resulting glycoproteins fold into their mature conformation on their way through the **ER** and **Golgi**.
HA and NA translated in the cytoplasm and modified in the ER/Golgi
4. The HA and NA are further transported through transport vesicles to the plasma membrane. They accumulate in the cell membrane at so-called lipid-drafts, where also the other components of the virus assemble
5. When sufficient HA and NA is present in the plasma membrane, the new virus can obtain an envelope derived from the plasma membrane by "**budding**", thereby leaving the cell
**NA** inactivates cellular receptors during escape from the infected cell. This prevents binding and subsequent re-infection of an already infected cell, thereby allowing the virus to be released from the infected cell in which it is produced and infect other cells.
27
Differences between poliovirus and influenza replication
Poliovirus:
- Replication in **cytoplasm**
- Makes RdRP during replication
- Vesicles for replication made in ER
- Do not bud and have no envelope
Influenza:
- Replication in **nucleus**
- Co-packaging of the RdRP in the virus particle
- Synthesis of viral glycoproteins in the ER
- "**Budding**" of progeny virions at the plasma membrane, during which the virus particles obtain a **lipid envelope**
28
Genome replication cycle of Influenza A virus
A virus particle that carries a **negative (-)** sense RNA genome first needs to transcribe its viral genome into a positive-stranded copy that can then serve as mRNA for protein synthesis.
Negative sense viruses need to co-occlude the viral **RNA-dependent RNA polymerase (RdRP)** in the virus particle in order to be able to:
- First make the **(+) strand RNA** from the (-) stranded viral RNA that then can be translated by ribosomes into proteins, such as more RdRP.
- The (+) stranded copies also serve as template to make new (-) stranded copies that can then be packaged into progeny virus particles.
The virus particle contains the (-) vRNA, and hence, introduce (-) strand RNA into the host cell, where a mRNA is generated from which proteins can be made. In a later stage, the (-) vRNA is reverse copied in a (+) strand RNA that is then used to make more (-) strand RNAs. Those (-) strand copies are incorporated into new virus particles.
The RNA genome is covered with proteins right after it is being synthesized (the blue dots) to protect the virus from decay. Hence, the name **Ribonucleoprotein (RNP)** complex for the influenza virus.
29
Influenza A virus: reassortment or “antigenic shift”
Influenza viruses use 2 mechanisms to adapt and escape from the immune defence system:
1. **Antigenic drift: Point mutagenesis**
--> Natural mutation over time: few amino acids per year due to mistakes
-->Relatively slow
--> Antigens drift away from what they originally were, and the immune system cannot recognize them any more
2. **Antigenic shift: re-assortment**
--> Exchange of its (8) *genome segments* during mixed infections, resulting in re-assorted new viruses
--> Shock-wise (step in evolution); causing pandemics
--> Two or more different strains of a viruses infect the same cell simultaneously and combine to form a new subtype having a mixture of the surface antigens of the two of more original strains
--> Many different possibilities
With influenza, mutations in the surface molecules of the virus help the organism evade the protective immunity that may have been obtained in a previous influenza season, making it necessary for individuals to get vaccinated every year.
30
HIV infection
Opportunistic infections are considered with AIDS-defining conditions
- More vulnerable for other type of infections
- Conditions related to a dysfunction immune system
- May carry oncoviruses, immunodeficiency
- CD4+ T-cells are killed by the virus
- Immune system goes up but eventually gets attacked by the virus
- Lysis of infected lymphocytes
- Lytic virus; lymphocytes lyse
31
Structure of a retrovirus
- **Lipid envelope glycoprotein SU** (surface) is the receptor-binding subunit, **TM** the membrane-anchored subunit: to attach to host cell receptors
--> **GP41/TM** makes sure that the proteins are embedded in the viral envelope
--> **GP120/SU**
--> Form trimeric spikes
- **RT: reverse transcriptase**
- **Integrase**
- **2 copies** of the viral RNA
--> Encapsulated by **nucleocapsid protein (NC)**
--> **Capsid coat** is surrounded by a lipid envelope
- **(+) strand RNA**: coded for proteins
32
Genome organization of HIV-1
- GAG = capsid protein
- Env= envelope protein
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The retroviral replication cycles
- **Reverse transcription** of the RNA genome into DNA (cytoplasmic)
--> Reverse transcription; reverse transcriptase, an RNA-dependent-DNA-polymerase
- **Integration** of the new DNA in the host chromosomal DNA by **integrase**
--> Integrated copy is called a “**pro-virus**”, with often a long nuclear latency period
--> mRNA can be made from the pro-virus, and the mRNA can make the viral proteins
1. Receptor mediated entry
2. Encapsidated viral **RNA** is converted into **dsDNA** by **reverse transcription** (in the cytoplasm)
3. dsDNA goes into **nucleus** and integrates with host chromosomal material
4. The pro-virus is **transcribed into RNA** (both mRNA and RNA) by the host’s **RNA polymerase II**
Retroviruses, such as HIV, have an RNA genome that must be reverse transcribed into DNA, which then is incorporated into the host cell genome. To convert RNA into DNA, retroviruses must contain genes that encode the virus-specific enzyme reverse transcriptase that transcribes an RNA template to DNA. Reverse transcription never occurs in uninfected host cells—the needed enzyme reverse transcriptase is only derived from the expression of viral genes within the infected host cells.
34
HIV genome composition
Structural proteins genes (incorporated in the virions):
- **Gag gene**: encodes for all internal structural proteins
--> Nucleoprotein, capsid protein, matrix protein
- **Pol gene**
--> For reverse transcriptase (RT)
- **Env gene**
--> For 2 envelope glycoproteins GP120 and GP41
- **Vpr gene**: transports the pre-integration complex (the DNA coy made in the cytoplasm) into the nucleus, where the process of viral integration into the host genome.
The **HIV provirus** is copied in a full-length mRNA copy by host derived **RNA polymerase II**. This full-length copy is then used to produce various **mRNAs** via **differential splicing**. These are then *translated* into **proteins**, which might be cleaved to obtain all functional HIV proteins.
Later in infection the **provirus DNA** is copied into **new viral genomic RNA**.
35
HIV-1 escape from the immune system
HIV mutates so rapidly, that when the immune system has developed antibodies, the HIV has already been mutated and escapes from these antibodies.
36
Recognition and binding to the cell receptor
For **recognition** of host cellular receptors, one or more surface proteins of the virus particle will have “**receptor-binding sites**” that are crucial for binding of the virus to the cell surface.
- Recognition of these (conserved) binding sites by the immune system will lead to production of **antibodies** that through their recognition of the receptor-binding sites will prevent these sites from binding to the receptor on the cell surface.
- As a consequence, these particular antibodies will neutralize the virus (**neutralizing antibodies**).
One or more viral surface proteins are involved in recognition and binding to the cell receptor.
- For viruses with a **protein coat** (such as picornaviruses) it is obvious that the coat proteins are responsible for receptor binding,
- For **enveloped viruses** (like influenza virus) **glycoproteins** in the membrane are involved in receptor binding.
*Initial stages of virus – cell interactions*:
- Plasma membrane fusion
- RNA-injection
- Endocytosis
--> Polio recognizes receptor and enters by endocytosis.
1. Interaction with attachment receptors and first conformational changes
2. Interaction with co-receptors/entry mediators and induction of further conformational changes
3. pH-independent fusion at the plasma membrane, or endocytosis and pH-dependent fusion/entry/uncoating
4. Released nucleocapsids and initiation of transcription
37
Viruses use (different) cellular receptors to enter the host cell
**Cellular receptors** are mostly **transmembrane proteins** (located at the plasma membrane, TMP) with an essential function for the (non-infected) host
- Viral cognition is based on specific amino acid sequences or folds in these proteins or certain carbohydrate side chains on TMP (glycoproteins)
--> Glycoprotein Heparan sulfate
- Makes it vulnerable for viral infection
38
There are 2 important host range determinants for specificity
- Host range
--> Can be narrow (limited to one host species) or very broad
- Tissue specificity (tissue tropism)
--> Foot-and-mouth disease virus (FMDV) can infect even-toed hoofed mammals
--> HIV-1 can only infect humans and this virus has a tissue tropism, which is restricted to T lymphocytes and macrophages.
39
Which viruses compete for the **same** receptor
- Coxsackievirus (a picornavirus) and Adenovirus (a large DNA virus) use the same receptor to enter particular cell types, and this receptor is therefore called CAR (Coxsackie and Adenovirus receptor).
- **CAR** is a transmembrane protein that belongs to the immunoglobulin superfamily.
40
Explain how viruses that can make use of several receptors can infect several tissues
- Being able to use more than one receptor can also be a way for a virus to expand its host range.
- The presence of a proper receptor is a first requirement for any virus to be able to infect a specific host and particular tissues in that host.
41
Poliovirus entry
The poliovirus receptor: **PVR**: human transmembrane protein
- In the human body, expressed in several tissues and cell types
- Explaining the tissue tropism and crucial host range determinant
The virus binds to a receptor on the host cell (receptor fits in ‘pocket/notch’ in virus). Interaction of the virus and the receptor facilitates an irreversible conformational change of the viral particle necessary for viral entry. The ‘pockets’ for binding in the virus are very tight and narrow; antibodies cannot enter these viral pockets.
42
Picornavirus entry
Different receptors are used by different types of virus. This discriminates on what type of hosts the virus can bind and infect.
*Uses several classes of receptors*:
- **Ig-like**: Single chain polypeptides
--> Domain 1 of each is involved in virus binding
--> ICAM-1, PVR, CAR
--> Immunoglobin superfamily
- **VLA-2**: 2 polypeptide chains
--> Echovirus
--> Integrin superfamily
43
HIV cell entry
Similar as Influenza.
*Steps in HIV cell entry*:
1. HIV binds via **SU (gp120)** to the **primary CD4 receptor** at the cell membrane
2. After binding of receptor and co-receptor the GP120 undergoes a conformational change
3. **TM (gp41)** achieves membrane fusion
Besides CD4, HIV also uses a **co-receptor**.
- For **macrophage infection** this is a protein called **CCR5**,
- For **T cell** infection this is **CXCR4**;
Both normally acting as “chemokine” receptors. The interaction of the virus with the co-receptor *precedes* the binding to the primary infection.
- HIV-1 *enters* the cell via **direct fusion** or **endocytosis**.
- Newly formed virus particles *leave* the cell via “**budding**”
- DCs in mucous membranes of genitals and blood-blood contact are regarded as the “main entre” of the virus
44
Tissue tropism of HIV-1
- CD4 is involves in the binding of CD4+ T cells to antigen-presenting cells.
--> Since CD4 is mostly present on the surface of T helper cells and macrophages, the tissue tropism of HIV-1 is mainly restricted to these blood stream located cells.
- It is clear that this **tissue tropism** explains the immune-deficiency syndrome after HIV-1 infection:
--> After infection most of the T helper cells will lyse.
-->HIV-1 infects dendritic cells, which occur in skin and mucous membranes.
45
HIV escape from the infected cell
1. **Nef-protein** removes CD4 receptor on Th-cells
2. **Cellular AP-2 clathrin adapter** recognizes this Nef-protein and leads CD4-nef to clathrin coated pits
3. CD4 receptor is *removed* by **clathrin-mediated endocytosis**
a. Cell has no longer CD4 receptors on the surface
b. Number of CD4-receptors drops
4. Newly made HIV particles can escape from the cell
