Virus assembly, maturation and exit Flashcards
What are packaging signals?
Viral genomes need to be distinguished from cellular DNA or RNA molecules where assembly takes place and this requires discrimination among similar nucleic acid molecules. Discrimination is the result of packaging signals in the viral genome and these are sequences that are necessary for the incorporation of nucleic acids into virions, genetically defined and are usually designated psi.
What are the mechanisms of virion assembly?
The virion structural units may assemble through:
- Association of individual monomeric proteins that are translated as separate components
- From large polyprotein subunits that are refolded after proteolytic processing
- With the assistance of viral or cellular chaperone proteins that facilitate correct folding of the virion structural proteins
- Using viral scaffold proteins that are not part of the mature virus particle but are used during assembly of immature virions to bring components together - then these scaffold proteins are removed
How do viruses assemble in the nucleus?
DNA viruses that replicate and accumulate their genome in the nucleus typically assemble in the nucleus however the exceptions are Hepadnaviruses and RNA viruses that replicate in the nucleus.
New DNA virus particles are usually released after breakdown of the cell due to the toxic effects of the viral proteins. Most DNA viruses assemble in the nucleus and to encapsidate a dsDNA genome, many viruses import their structural proteins into the nucleus through the nuclear pore. This poses an issue because these proteins are being translated in the cytoplasm and so somehow they must go back to the nucleus. This is achieved by nuclear localisation signals and they mediate protein transport from the cytoplasm through the nuclear pore.
What are nuclear localisation signals?
Many viral proteins contain a region of protein sequence that contains a nuclear localisation signal (NLS) that binds to nuclear transport proteins. There are different types of nuclear localisation signals, but a common feature is the high content of basic amino acids. Proteins that have this are able to transit through the nuclear pore.
What is involved in protein translation?
Protein translation can proceed on free ribosomes in the cytoplasm and membrane bound glycoproteins are translated on ribosomes of the ER.
What are packaging signals of the DNA genome?
This is important for packaging the new viral DNA into new infectious particles and they overlap the enhancer region. The enhancer sequences are those that interact with cellular transcription factors and these promote the expression of the genes.
What are some examples of packaging signals of DNA viruses?
Adenovirus:
- Packaging signal is near the left inverted repeat and origin
- The signal is complex as they are a set of repeated sequences that overlap with enhancers that stimulate transcription
- The structure is recognised by the viral protein IV2a (also a transcription factor)
SV40 virus:
- The Ori site (origin of replication) is a busy site as it also contains the transcription units but overlapping this is also the packaging signal.
What are some examples of packaging signals of RNA viruses and what is an example?
An example is HIV virus.
- The genome of the packaging signal has self-complementarity forming these three or four stem loop structures (SL1, 2, 3 and 4).
- It is stem loop 1 that contains a sequence at the tip that has a lot of self-complementarity and they form these kissing loop structures (palindromic).
- The two adjacent RNAs of the diploid genome to come together and assemble into a loose interaction.
- The loose interaction brings and binds the two monomeric RNAs into a dimeric RNA structure.
- There is also the dimer initiation sequence and dimer linkage sequence (DIS and DLS). These are sequences that have the capacity to interact with the nucleocapsid protein (this binds directly onto the RNA genome). It has a zinc finger binding domain that allows it to assemble onto the dimer linkage sequences forming a structure that is stabilised dimer of these RNAs.
- The HIV packaging signal is placed within spliced regions - this ensures that no mRNAs are packgaged and only the genomic RNA - NC of Gag mediates selective encapsidation of genomic RNA during assembly - central region of the NC binds RNAs with psi sequences, however will only recognise the dimeric RNA structure
What are examples of individual monomeric proteins being translated as separate components?
The SV40 virus spliced mRNAs translate individual monomeric proteins that assemble into the individual structural components of the virion protein shell.
The adenovirus monomeric proteins that assemble into the penton units first assemble as homo-multimers that are then assembled into a complete unit.
What are examples of large polyprotein subunits that are refolded after proteolytic processing? With poliovirus, how is the capsid formed after processing?
Poliovirus.
Many viruses require correct folding and proteolytic cleavage of viral polyproteins to produce an individual structural unit for capsid assembly. The 5S structural unit of the poliovirus form after cleavage by the 3CDpro viral protease that cleaves all of the subunits, except cleaving VP0 into the VP2 and VP4 subunits.
The capsid/nucleocapsid is formed by assembly and the electrostatic complementarity of the interface between the subunits promotes similar associations between proteins. The strength of the shaking is analogous to the temperature of the molecular environment and if it is too hot then the virus will break apart.
What are examples of the chaperone proteins?
Adenovirus hexon units are made up of trimers of adenoviral protein II and these are assembled onto the surface with the assistance of a viral chaperone protein known as L4. This does not become part of the virion, but new virus particles cannot form without it.
Why are hepadnaviruses the exception to assembly in the nucleus? What about RNA viruses that replicate in the nucleus?
These have an RNA pre-genome that assembles in the cytoplasm - an example is for the hepatitis B virus. The influenza virus and the retroviruses have envelopes and assemble at the plasma membrane.
How does the SV40 virus and adenovirus import their structural proteins into the nucleus?
These are DNA viruses that assemble in the nucleus. SV40 polyomavirus VP1 + VP2/3 have nuclear localisation signals that direct the protein into the nucleus.
The adenovirus hexon proteins lack a NLS but the chaperone protein L4 mediates nuclear import.
How are herpesvirus genomes encapsidated, assembled and matured?
Herpes virus genome replication produces concatamers with head to tail copies of viral genome. The HSV 1 and 2 packaging signals, pac1 and pac2, are needed for recognition of viral DNA and cleavage within DR1. Upon recognition of both ends of the genome, it is cleaved and encapsidated.
Seeing as it is a DNA virus, it only assembles in the nucleus and once the encapsulated genome within the capsid is assembled, it then processes through several layers of membranous structures where other viral proteins are assembled into a structure known as the tegument layer. Many of these are processed, trimmed, modified and repackaged into the infectious particle that forms and buds out of the surface of the plasma membrane following this complex transport through the ER and trans-golgi network. Transport out is mediated via envelopment/de-envelopment of the nuclear membrane.
exit via exocytosis.
What happens to the protein during translation at the ER?
Ribosome translating proteins, which have an uninterrupted sequence of about 20 hydrophobic amino acids at their beginning, attach to the ER membranes and the hydrophobic signal sequence directs the protein into the lumen of the ER. Once the hydrophobic leader sequence completes its transition, the signal peptidase cleaves it off and the remainder of the protein is secreted or exported into the lumen of the ER.
