Lecture 3+4 Virus infection

Question 1

Baltimore classification of viruses

Answer 1

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.

Question 2

Group I viruses

Answer 2

dsDNA

• +/- DNA –> + mRNA

Example: herpes simplex/adenovirus

Question 3

Group II viruses

Answer 3

(+) ssDNA

• + DNA –> +/- DNA –> + mRNA

Example: parvovirus

Question 4

Group III viruses

Answer 4

dsRNA

• +/- RNA –> + mRNA

Examples: reovirus/rotavirus

Question 5

Group IV viruses

Answer 5

(+) ssRNA.

• + RNA –> - RNA –> + mRNA

Example: poliovirus

Question 6

Group V viruses

Answer 6

(-) 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

Question 7

Group VI viruses

Answer 7

(+) ssRNA (RT)

• + RNA –> - DNA –> +/- DNA –> + mRNA

It must be converted, using the enzyme reverse transcriptase, to dsDNA.

Example: HIV/retrovirus

Question 8

Group VII viruses

Answer 8

(+/-) 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

Question 9

Virions

Answer 9

Virus particles

Question 10

Positive-strand RNA virus

Answer 10

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

Question 11

Negative-strand RNA virus

Answer 11

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

Question 12

Poliovirus

Answer 12

Positive stranded, non-segmented RNA virus.

Primarily infection in the GI tract. It can affect the nervous system.

Question 13

Influenza A virus

Answer 13

Negative stranded segmented RNA virus

Question 14

HIV

Answer 14

Retrovirus: copying its RNA genome into integrated DNA prior to protein expression

Question 15

Linear RNA

Answer 15

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.

Question 16

How are viral proteins being made

Answer 16

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:

1. They may have a single ORF encoding a large polyprotein, which is later cleaved in functional proteins (e.g. poliovirus).
2. Some RNA viruses have segmented genomes, where every RNA molecule carries the information for one protein (e.g. influenza virus).
3. To make sub-genomic mRNAs (using a full length (-) RNA molecule as template) to express all genes (e.g. Chikungunya virus).
4. Making use of differential splicing (e.g. HIV) resulting in individual mRNA molecules for the various proteins

Combinations of these strategies may also occur.

Question 17

Gene functions of the poliovirus

Answer 17

• 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

Question 18

Structural proteins

Answer 18

Proteins of the viral capsid

Question 19

Non-structural proteins

Answer 19

Not being part of the virus particle, but only formed and function inside the infected cell. They are needed for virus replication.

Question 20

Genome replication of poliovirus

Answer 20

Replication of the polioviral RNA, (making new genome copies), occurs in membrane vesicles in the cytoplasm induced by this virus.

1. Binding of the poliovirus coat protein VP1 to host poliovirus cell receptors (PVR) on the cell surface (transmembrane protein) mediates endocytosis
2. 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
3. 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
4. Replication via dsRNA occurs in viral factories made of membrane vesicles derived from the ER (copy)
5. The dsRNA genome is transcribed/replicated to produce mRNA and new ssRNA(+) genomes
6. Polyprotein is cleaved into the smaller, mature viral proteins by 2A and 3B
7. New viral RNA is packaged into preassembled procapsids (capsid formation)
8. Cell lysis (bursts open) and millions of virus particles release
9. 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.
10. The new (-) strand then serves as template to make more (+) strands.
11. These new (+) strands can either be translated or become packaged in progeny virus particles.

Question 21

What are the final products of translation of the poliovirus

Answer 21

• 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).

Question 22

Which three intermediate cleavage products are generated in the translation of the poliovirus

Answer 22

• 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

Question 23

Influenza virus A

Answer 23

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

Question 24

The schematic structure of influenza A virus

Answer 24

• 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.

Question 25

Indicate types of influenza A virus

Answer 25

• 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.

Question 26

Replication cycle of Infuenza virus A

Answer 26

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.

Question 27

Differences between poliovirus and influenza replication

Answer 27

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

Question 28

Genome replication cycle of Influenza A virus

Answer 28

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.

Question 29

Influenza A virus: reassortment or “antigenic shift”

Answer 29

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.

Question 30

HIV infection

Answer 30

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

Question 31

Structure of a retrovirus

Answer 31

• 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

Question 32

Genome organization of HIV-1

Answer 32

• GAG = capsid protein
• Env= envelope protein

Question 33

The retroviral replication cycles

Answer 33

• 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.

Question 34

HIV genome composition

Answer 34

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.

Question 35

HIV-1 escape from the immune system

Answer 35

HIV mutates so rapidly, that when the immune system has developed antibodies, the HIV has already been mutated and escapes from these antibodies.

Question 36

Recognition and binding to the cell receptor

Answer 36

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

Question 37

Viruses use (different) cellular receptors to enter the host cell

Answer 37

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

Question 38

There are 2 important host range determinants for specificity

Answer 38

• 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.

Question 39

Which viruses compete for the same receptor

Answer 39

• 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.

Question 40

Explain how viruses that can make use of several receptors can infect several tissues

Answer 40

• 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.

Question 41

Poliovirus entry

Answer 41

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.

Question 42

Picornavirus entry

Answer 42

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

Question 43

HIV cell entry

Answer 43

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

Question 44

Tissue tropism of HIV-1

Answer 44

• 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.

Question 45

HIV escape from the infected cell

Answer 45

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