1. Virus Assembly

2. Final Stages of Infection Cycle
Assembly
Exit from cell

3. Assembly initiates when threshold quantities of progeny virus genomes and structural proteins have accumulated in the infected cell
Nucleocapsid no envelope.
Nucleocapsid with lipid envelope

4. Nucleocapsid Assembly
Helical - coating the genome with multiple copies of a protein

5. Icosahedral - construction of an empty protein shell known as a procapsid, or a prohead in the case of a tailed bacteriophage. The procapsid is filled with a copy of the virus genome.

6. ‘How are virus genomes selected from all the cell and virus nucleic acids?’
Cell nucleic acids are not packaged.
For some viruses it has been shown that this is achieved through the recognition by a virus protein of a specific virus genome sequence, known as a packaging signal.
In single-stranded genomes the packaging signal is within a region of secondary structure.
The packaging signal must be present only in the strand to be packaged.
It is important that some copies of the virus genome are not packaged: those that are serving as templates

7. Genome Packaging
Virus genomes are packaged into small volumes, which means that repulsion between the negative charges on their phosphate groups must be overcome.
Viruses have evolved a number of mechanisms:
packaging basic proteins, which are positively charged, along with the genome. E.g some ssRNA viruses (e.g. rhabdoviruses, influenza viruses and retroviruses)
Some dsDNA viruses (e.g. Adenoviruses and baculoviruses) have a basic protein closely associated with the genome.
But papillomaviruses and polyomaviruses coat their DNA with host cell histones, the basic proteins associated with the DNA in eukaryotic cells.
Some viruses package other positively charged materials, such as polyamines and cations.
dsDNA of large icosahedral viruses such as herpesviruses and the tailed phages (T4) is packed so tightly that the pressure within the capsid is about ten times greater than the pressure inside a champagne bottle.

8. Assembly Mechanisms
Self assembly – simple viruses assemble from components into virions e.g tobacco mosaic virus (TMV).
Directed assembly – complex viruses that do not assemble in test tube need additional help need host cell

9. Formation of virion membranes
Enveloped virions acquire their membrane envelopes by one of two mechanisms;
either they modify a host cell membrane and then nucleocapsids bud through it,
or the virus directs synthesis of new membrane, which forms around the nucleocapsids

10. Budding through cell membranes
Most enveloped viruses acquire their envelopes by budding through a membrane of the host cell
For viruses with eukaryotic hosts this membrane is often the plasma membrane.
Most retroviruses and rhabdoviruses acquire their envelopes in this way.
Regions of membrane through which budding will occur become modified by the insertion of one or more species of virus protein, the vast majority of which are glycoproteins.

12. Budding of virions involves interaction between the cytoplasmic tail of a virus glycoprotein in the membrane and another virus protein.
In a number of virus groups, including paramyxoviruses and rhabdoviruses, this protein is the M (membrane, matrix) protein
M1 protein of influenza A virus and the MA (matrix) domain of the retrovirus Gag protein

13. Polarized Budding
Viruses that bud from the cell do so from particular regions of the plasma membrane.
Body surfaces, such as the respiratory tract, are lined with epithelial cells that are polarized.
Each cell has an apical (outer) and a basolateral (inner) surface and many viruses bud preferentially from one of these.
Influenza A virus buds almost exclusively from the apical surface, while vesicular stomatitis virus buds almost exclusively from the basolateral surface.
Experiments in which the envelope protein genes of these viruses were expressed in cells indicated that each protein has a signal that targets it to the surface from which the virus buds.

14. The release of influenza virus particles from cells requires the activity of a virion enzyme: a neuraminidase
At budding sites cleavage of neuraminic acid, the substrate for the enzyme, is essential for the release of virions.
Some anti-influenza virus drugs have been developed that act by inhibiting the neuraminidase

15. Budding from other cell membranes
Some enveloped viruses bud from cell membranes other than the plasma membrane.
Herpesvirus nucleocapsids are constructed in the nucleus and begin their journey to the cytoplasm by budding through the inner membrane of the nuclear envelope; the envelope of the mature virion is acquired in the cytoplasm by budding into vesicles derived from the Golgi complex
Hepadnaviruses bud into a membrane compartment between the endoplasmic reticulum and the Golgi complex.

16. De novo synthesis of viral membranes .
A minority of viruses direct the synthesis of lipid membrane late in the replication cycle. E.g poxviruses
Baculoviruses produce two types of enveloped virion during their replication:-
One type of virion has the function of spreading the infection to other cells within the host, and this virion acquires its envelope by budding from the plasma membrane.
The other type of virion has the function of infecting new host individuals through the formation of occlusion bodies.

17. General consideration
Enveloped viruses – nucleus
Enveloped viruses – cytosol
Non-enveloped viruses – nucleus
Non-enveloped viruses - cytosol

19. Steps involved in viral assembly
Viral protein and nucleotide synthesis
Viral protein and nucleotide trafficking
Encapsidate viral nucleotides to assemble viral core
Envelopment of virus and retention of viral glycoproteins (for enveloped viruses)
Viral release: budding and lysis of cells
Virion maturation (proteolytic process)

20. Intimate involvement of cellular machinery in viral assembly
Cellular polymerase or a virus-encoded polymerase to viral genome replication
Cell translational machinery for viral protein synthesis
Cellular sorting machinery for viral components trafficking and releasing
Cellular proteases, membranes, glycosylation machinery, kinases and chaperon proteins for protein maturation

21. Protein localization in a mammalian cells

22. Budding sites for different viruses

23. Assembly of influenza virus

24. Issues in influenza virus assembly
Packaging a segmented genome: random versus precision
Export of nucleocapsids from the nucleus to the cytoplasm: the role of M1 and NS2
The biogenesis of HA proteins: the role of cellular endopeptidase and M2
The role of NA and monoubiquitination in virion budding from cell surface

25. Assembly of HIV

26. Important issues in HIV assembly
Export of viral RNA: the role of rev protein
Biosynthesis of envelope proteins: the role of endopeptidases
Egress of virion: the role of late domain of gag p6 and gag MA proteins
Downregulation of CD4: the role of nef, vpu, and gp120 proteins
The role of exosome and lipid raft
The maturation of virion: the role of viral protease

27. Summary
Our knowledge in viral assembly is incomplete and fragmented. Some are still very descriptive.
Viral assembly involves intimate interactions between viral and host factors
Understanding these interactions will shed light on biological processes of cells
Understanding these interactions will help to develop new therapeutic intervention strategies