Virus facts

Question 1

Polyomaviruses structure

Answer 1

• Small DNA viruses that drive cells into S phase
• Associated with post organ transplant kidney malfunction and merkel cell carcinoma
• Smallest dsDNA viruses that infect animal cells
• Non-enveloped spherical virions with 4- to 5-kbp circular dsDNA genome

Question 2

HPV structure

Answer 2

Spherical virions with circular dsDNA genome 6-8.5kbp

Question 3

Poxviruses structure

Answer 3

• Largest dsDNA virus that infect humans
• Linear genome is composed of 194 kbp of dsDNA with inverted terminal repeats that form covalently closed hairpin termini
• Enveloped, brick-shaped or ovoid virion

Question 4

Flaviviruses structure

Answer 4

• Enveloped, spherical virions, 10-12kb, +ssRNA genomes
• 5’ cap similar to host mRNA
• Lack 3’ poly A tail, instead 3’ UTR folds into secondary structures with several stem loops

Question 5

Picornaviruses

Answer 5

lack 5’ cap and itilise VPg protein and IRES

Question 6

Baltimore class V

Answer 6

-ssRNA that may be segmented or non segmented

Question 7

Mononegavirales

Answer 7

• ss(-)RNA viruses with non segmented genomes
• Rabies and measles
• After uncoating, the (-) RNA remains associated with nucleocapsid proteins (NPs), other viral proteins (including RdRp) and associated factors needed for RNA synthesis
• This complex, including the genome, is called viral ribonucleoprotein complex (vRNP)
• RdRp becomes active as a transcriptase, using vRNP as a template to synthesise viral mRNAs
• When levels of NP reach a threshold, anti genomes are synthesised and new genomes which are packaged into new virions during maturation

Question 8

Orthomyxoviridae

Answer 8

• Influenza
• 8 genome segments each bound to NP and PA, PB1 and PB2
• The 3’ end and 5’ end contain secondary structures

Question 9

Reoviridae / rotavirus

Answer 9

• Non enveloped, icosahedral virion with triple capsid structure
• Segmented linear dsRNA genome with 11 segments coding for 12 proteins
• Each segment has 5’ cap, no polyA tail

Question 10

Rotavirus site of viral dsRNA synthesis

Answer 10

Viroplasm

Question 11

Rotavirus site of early transcription of dsRNA genoma by RdRp

Answer 11

Inside double layered particle

Question 12

HIV part 1

Answer 12

• HIV-1 infects CD4+ T cells via binding of surface gp120 to CD4 receptor
• This triggers a conformational change that initiates gp120 binding to a co-receptor CCR5/CXCR4
• This triggers rearrangement of the gp120-gp41 complex such that the fusion peptide of gp41 inserts into the plasma membrane and the two fuse.
• The HIV nucleocapsid releases into the cytoplasm, releasing the viral RNA genome and enzymes (RT, integrase, protease) into the cytoplasm
• RT converts ssRNA into dsDNA which is transported into the nucleus where it integrates into a host chromosome using viral integrase

Question 13

HIV part 2

Answer 13

• Viral genes are transcribed by host pol II to produce eukaryotic mRNA and mRNA is transported to cytoplasm where translation occurs
• Some proteins enter the nucleus to affect gene expression
• Major structural proteins that form the internal parts of the virion are synthesised as polyproteins Gag, Gag-pol and Env.
• Protease cleaves Gag/Gag-pol polyproteins to produce infectious virus particles with a morphologically distinct core
• HIV-1 nef/vpu mediate translocation of cell-surface CD4 to the cytoplasm/lysosol allowing new virions to leave the cell

Question 14

Reovirus replication cycle

Answer 14

• Reoviruses ( eg. Rotavirus) are unusual in that the infecting virion remains largely intact inside the host cell for all or some of the gene expression stages.
• Have segmented dsRNA genomes enclosed by three layers of protein.
• These naked viruses are internalized by endocytosis after which the outer capsid disassembles and the double-layered particle is present in the cytoplasm.
• The double-layered particle remains intact and serves as the first site of virus mRNA production, when enzymes in the core use the genomic RNA as a template to synthesize, cap, and tail viral mRNAs.

Question 15

Reovirus structure

Answer 15

The genome segments are surrounded by the inner capsid, a second a double layered intermediate capsid particle, and third layer called the outer capsid.

Question 16

First 5 steps of reverse transcription in HIV-1

Answer 16

1. (First strand (-) cDNA synthesis is initiated in a 5’ direction.
2. RNase H activity degrades the small fragment of complimentary template RNA
3. This allows for the first jump to occur. Complimentary R
   (R’) within the newly synthesised (-)cDNA is complimentary to R in the 3’ end of the RNA template, allowing for RT extension
4. (-)cDNA extension occurs towards the 5’ end of the template
5. With RNase H activity degrading the template RNA, leaving behind only the RNA complimentary to PPT

Question 17

Steps 6-8 of reverse transcription in HIV-1

Answer 17

6. PPT’ provides a primer for the synthesis of ds CDNA (+), complimentary to the (-)cDNA
7. RNase H activity removes all remaining RNA template, allowing for the second jump, with PBS from the second
   strand complimenting the PBS’ of the 5’ end of the (-)cDNA template
8. Result is dsDNA with long terminal repeats (LTRs) at both ends, containing U3, R and U5

Question 18

Influenza virus replication cycle steps 1-6

Answer 18

1. Virus attaches to its receptor (sialic acids (SA, N-acetylneuraminic acid) on the surface of the host cell.
2. The virus is internalised by endocytosis during the penetration step.
3. During uncoating, the (ss -RNA) genome segments and vRNPs are released into the cytoplasm
4. And are transported to the nucleus
5. The influenza genome is copied to make a double stranded template
6. ds template can be used for production of mRNA and new genomes.

Question 19

Influenza virus replication cycle steps 7-11

Answer 19

7. The mRNAs are exported to the cytoplasm
8. mRNAs are translated to make IV proteins.
9. Some influenza proteins reenter the nucleus to assemble with new genome segments, whereas others assemble at sites of future budding
10. The vRNPs assemble with other component parts of the virus.
11. The virus exits the host cell through budding, assisted by a viral enzyme (the neuraminidase, NA) that degrades the host surface sugars (sialic acids (SA, N acetylneuraminic acid) that would otherwise tether the virus to the cell surface.

Question 20

Acquisition of envelope by IVA and virus budding

Answer 20

• Viral proteins are synthesized in the cytoplasm and on the endoplasmic reticulum.
• Viral proteins that will become part of new vRNPs must be trafficked to the nucleus while the viral envelope proteins traffic to the plasma membrane
• New vRNPs are synthesized in the nucleus and are then exported to the cytoplasm. They travel along the microtubule network at the periphery of the cell, bundling along the way.
• Eight different vRNPs gather under a patch of plasma membrane containing matrix and spike proteins
• The new virion buds away from the surface of the host cell.

Question 21

First step of influenza transcription

Answer 21

• IVA produces 10 seperate mRNAS with 5’ methylated caps, however doesn’t encode enzymes to synthesises these caps
• First step is cap snatching: the cap binding motif in PB2 protein (within vRNP) binds to host mRNA before it leaves the nucleus
• After PB2 binds to host mRNA, the PA protein cleaves the host mRNA between 9-15 nucleotides downstream of the cap
• Cleaved mRNA then serves as a primer for synthesis of mRNA that is complimentary to the RNA in the vRNP
• RdRp acts in cis to elongate the capped primer using vRNP as template whilst remaining attached to the 5’ end of template genomic RNa
• mRNA is released and the vRNP returns to original config

Question 22

Adenovirus entry and movement in cells

Answer 22

• After attachment, adenovirus is internalized through clathrin-coated pits and enters the endosome system.
• The virus spike proteins dissociate from the rest of the capsid early after internalization.
• Acidification of the late endosome releases several proteins including protein VI, which is cleaved by a viral protease activated by the chemical conditions in the maturing endosome.
• Protein VI ultimately causes lysis of the endosomal membrane and the release of the partially disassembled adenovirus capsid into the cytoplasm.
• In an extended penetration and uncoating stage that can last 40–60 min, the capsid moves along the microtubule cytoskeleton (via Dynein), ultimately reaching a nuclear pore complex.
• Docking with the nuclear pore complex causes conformational changes in both the capsid and the nuclear pore complex, which together result in the release of virus DNA into the nucleoplasm