6 - Virus Assembly, Release and Maturation

Question 1

Viral assembly

Answer 1

• Refers specifically to the assembly of capsids with genomes and so is universal to all viruses.
• Enveloped viruses must also acquire a membrane during assembly

Question 2

Pathway of virus particle assembly and release

Answer 2

• Formation of individual structural units of the protein shell from one or several viral proteins
• Assembly of the protein shell by appropriate interactions among structural units
• Selective packaging of the nucleic acid genome and other essential virion compounds
• Acquisition of an envelope (not all)
• Release from host cell
• Maturation of virus particles

Question 3

Structural units that are often the first assembly intermediates

Answer 3

Homo or hetero-oligomers of viral structural proteins from which virus particles are built

Question 4

Typical sites of assembly in eukaryotic viruses and example for each

Answer 4

• Cytoplasm (bacteriophages)
• Nucleus (Adenovirus)
• Nucleus and cellular membranes (herpes and influenza)
• ER (coronavirus)
• Plasma membrane (retroviruses)

Question 5

Importins

Answer 5

Cellular proteins that transport cargo proteins from the cytoplasm to the nucleus, aided by nuclear localization sequences (NLS)

Question 6

Assembly within the nucleus

Answer 6

• Viral proteins destined for the nucleus are synthesized by cytoplasmic polyribosomes, engage with the cytoplasmic face of the nuclear pore complex and are translocated into the nucleus by the protein import machinery of the host cell.
• E.g. adenoviruses papillomaviruses, and polyomaviruses

Question 7

Virus assembly from constituent parts

Answer 7

Assembly from individual protein molecules (e.g. simian virus) or a polyprotein precursor (e.g. poliovirus) or chaperone assisted assembly (e.g. adenovirus)

Question 8

Assembly from individual protein molecules

Answer 8

• The assembly reaction is the result of specific interactions among the proteins that form structural units.
• These assembly reactions are driven in a forward direction by the high concentrations of protein subunits synthesized in
  infected cells

Question 9

Assembly from a polyprotein precursor

Answer 9

• Illustrated with the poliovirus polyprotein that contains the four proteins that form the heteromeric structural unit.
• These proteins are synthesized as part of the single polyprotein precursor from which all viral proteins are produced by proteolytic processing including the viral-encoded protease.

Question 10

Chaperone assisted assembly

Answer 10

Some structural units are assembled only with the assistance of viral chaperones, such as the adenoviral L4 100-kDa protein, which is required for formation of the hexon trimer from the protein II monomer.

Question 11

Chaperones

Answer 11

• Specialised proteins that facilitate the folding of other proteins by preventing improper, nonspecific associations among sticky patches exposed on newly synthesized proteins
• Abundant in all cells, and some accumulate to concentrations greater than ribosomes.
• Genomes of several viruses encode proteins with chaperone activity, some with sequences and functions homologous to
  those of cellular proteins

Question 12

Scaffolds

Answer 12

• Proteins that are needed for parts of the virus to assemble but are not found in the mature virion.
• Accurate assembly of some large icosahedral protein shells (e.g. adenoviruses and herpesviruses) is mediated by proteins that are not components of mature virus particles
• E.g. precursor of the herpes simplex virus 1 VP22a protein.

Question 13

Two strategies of capsids associating with genomes

Answer 13

• Concerted assembly
• Sequential assembly

Question 14

Concerted assembly

Answer 14

• The capsomers and genome come together during genome synthesis assembly and there is no separate empty capsid to be filled
• The nucleocapsid protein encapsidates new genomes while they are being synthesized. - If the virus is enveloped, a later
  assembly step leads to acquisition of a matrix and envelope surrounding the helical nucleocapsid.

Question 15

Example of concerted assembly

Answer 15

• Rabies virus and Ebola virus have small (−) RNA genomes that are associated with nucleocapsid
  protein N, forming viral ribonuclear proteins (vRNPs)
• The nucleocapsid N protein is required during genome replication, and the new genomic RNA is encapsidated into RNP during RNA synthesis.
• Influenza is also a (−) RNA virus that assembles new helical influenza vRNP during RNA synthesis.

Question 16

Concerted assembly of influenza virus RNPs

Answer 16

• The first stage of influenza assembly is for the PA, PB1, PB2, and NP proteins to form vRNP on new negative strands of RNA through concerted assembly
• For influenza virus, vRNPs arise from cRNP templates and the genomic vRNPs are packaged into helical structures during genome synthesis
• vRNPs must be packaged selectively with viral envelope, not cRNPs
• This is dependent on interactions with PA, PB1, and PB2 and the two RNA molecules
• These interactions mean vRNP are competent for export out of the nucleus, whereas cRNPs are retained in the nucleus
• Once exported, sequences near the 5ʹ and 3ʹ ends of the virus genome, which are conserved in all eight genome segments, target the vRNPs for packaging

Question 17

vRNP

Answer 17

viral ribonuclear protein

Question 18

cRNP

Answer 18

copy ribonuclear protein

Question 19

Sequential assembly

Answer 19

During sequential assembly, regular groups of capsomers such as pentamers or hexamers assemble first and then assemble together to make a hollow shell or procapsid.

Question 20

Assembly of HSV1 nucleocapsid

Answer 20

• Requires the scaffolding protein pre-VP22a
• Self-association of pre-VP22a stimulates binding to VP5, the
  protein that forms structural units of capsid
• V5 interaction with scaffolding protein guides and regulates VP5 self-assembly
• Omission of the scaffolding leads to production of partial and deformed nucleocapsid shells
• Once nucleocapsids have assembled, scaffolding proteins must be discarded, so that viral genomes can be accommodated

Question 21

Virion protease VP24

Answer 21

• Essential for DNA encapsidation
• This protein is incorporated into the assembling nucleocapsid as a precursor
• VP24 cleaves the scaffolding protein to remove a short C-terminal sequence that is required for binding to VP5, to disengage scaffolding from structural proteins, once assembly of the nucleocapsid is complete.
• The VP24 protease also degrades the scaffolding protein so that encapsidation of the genome can begin.

Question 22

Adenovirus L1 proteins

Answer 22

• Form structures that support assembly
• L1 decreases in concentration as assembly proceeds

Question 23

Viral L3 protease

Answer 23

Completes assembly by cleaving precursor proteins, resulting in a mature, infectious virus

Question 24

Localisation or viral proteins to plasma membrane

Answer 24

• Viral envelope glycoproteins are
  cotranslationally translocated into the ER lumen and folded and assembled within that
  compartment.
• They travel via transport vesicles to and through the Golgi apparatus and then to the
  plasma membrane.
• The internal proteins of the virus particle and the genome are also directed to plasma membrane sites of assembly

Question 25

Glycosylation of viral proteins

Answer 25

- May alter antigenicity - IV HA evolves at the highest rate compared to other influenza viral proteins - Presence and acquisition of N-linked glycosylation sites on HA plays a crucial role in immune evasion - Number of glycosylation sites on HAs appears to derive from a balance between functionality and immune evasion

Question 26

Two pathways of viruses acquiring envelopes

Answer 26

1.Assembly of the capsid and genome occurs first. Acquisition of the envelope occurs next: the assembled capsid interacts with a cellular membrane that also contains virus spike proteins and pinches off (e.g. influenza virus) 2. Formation of internal structures of the virion occur in coordination with the envelope eg. HIV

Question 27

Influenza virus assembly

Answer 27

- IV 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 28

Final step in IV release

Answer 28

Viral NA removes sialic acids from both cellular receptors and from newly synthesized HA and NA on nascent virions, which have been sialylated as part of the glycosylation processes within the host cell

Question 29

Neuraminidase inhibitors

Answer 29

Prevent virions from being released from the surface of infected cells

Question 30

HIV1 example of concerted viral assembly

Answer 30

- HIV virion consists of the Env proteins SU and TM, followed by MA matrix protein. - Inside of the MA ring, there is a cone-shaped capsid made of the CA protein. - The diploid RNA genome closely associated with the NC protein inside the CA. - The virion contains the enzymes protease, reverse transcriptase, and integrase, as well as the p6 protein - HIV virions also contain a specific tRNA molecule annealed to the RNA genomes - Virion also contains the accessory proteins Vif, Vpr, Vpx, and Nef

Question 31

HIV Gag and Gag-Pol polyproteins

Answer 31

Give rise to proteins MA, CA, NC, p6, protease, reverse transcriptase, and integrase after proteolysis

Question 32

Maturation

Answer 32

Any irreversible process that occurs during or after viral release that takes the assembled virus from a noninfectious to an infectious state

Question 33

HIV-1 protease

Answer 33

Cleaves gag and gag-pol polyproteins to produce infectious virus particles with a morphologically distinct core

Question 34

HIV-1 nef

Answer 34

Recruits cell-surface CD4 into the endocytic pathway where it is sorted into lysosomes and degraded

Question 35

HIV-1 vpu

Answer 35

Mediates translocation of cytoplasmic CD4 in the ER into the cytosol where it is ubiquinated and enters the proteosome to be degraded

Question 36

Release of virus from animal cells

Answer 36

Can occur by lysis or budding

Question 37

Adenovirus death protein

Answer 37

- Transmembrane protein that can be found in the nuclear membrane, endoplasmic reticulum, and Golgi apparatus - Kills cells when it accumulates during late stages of infection and causes lysis

Question 38

When do enveloped viruses acquire their envelopes

Answer 38

Either after the internal parts of the virion have assembled, (e.g. influenza virus) or during the process of assembling the internal components of the virion (e.g. HIV)