Virus Assembly

Final Stages of Infection Cycle Assembly Exit from cell

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

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

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.

‘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

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.

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

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

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.

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

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.

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

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.

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.

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

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)

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

Protein localization in a mammalian cells

Budding sites for different viruses

Assembly of influenza virus

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

Assembly of HIV

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

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