Maturation, assembly, and release

As proteins and  new  genomes are  synthesized in the  infected cell they  are  channeled to various locations for virion  assembly and  egress  (release). Depending on the virus, capsids assemble in the nucleus or the cytoplasm, either building around the genome molecule, or packaging it as a final maturation step following capsid assembly. Genome and capsid are released from cells by budding through a cellular membrane, or by caus- ing cell lysis. Enveloped viruses may bud through the nuclear membrane, plasma mem- brane or even the endoplasmic reticulum; prior to budding the virus-specific envelope glycoproteins have been laid down in the membrane.

The steps of capsid assembly vary according to the complexity of the mature capsid. Simple capsids such as those of poliovirus and HPV assemble through the association of structural proteins into units known as capsomers. The structural proteins of poliovirus ( VP0, VP1, and  VP3) first bind together in trimers that then associate as pentamers;  12 pentamers form a complete but immature icosahedral capsid. Using an unknown mech- anism that may in part depend on the covalently attached VPg protein, genomic RNA is sequestered into the capsid and the final maturation step, cleavage of VP0 into VP2 and VP4, seals the virion.  The HPV capsid is simpler again, comprising only two proteins. Pentamers of the major structural protein L1 form the major capsid structure, along with a few molecules of the minor protein L2, thought to locate at the vertices of the icosahe- dron. Packaging of the viral genome appears to involve the L2 and E2 proteins, although E2 is not found in the virion. Both poliovirus and HPV exit infected cells by lysis, releasing several hundreds of progeny virions.

Mature HSV capsids have several structural proteins and are assembled in the nucleus, assisted by two scaffolding proteins, UL26 and UL26.5, which associate with the major capsid protein (VP5) in a double-shelled procapsid structure. UL26 is a protease that cleaves the scaffolding proteins that exit the structure leaving an empty, immature icosa- hedron formed predominantly of VP5. Genome packaging is an elegant process whereby the long concatemers of replicated viral genomic DNA must be cleaved by an endonuclease at a precise point, to release individual genome molecules that can be packaged. The genome enters the immature capsid at one open corner, which is then sealed by a cluster of UL6 proteins. Several alternative paths for egress of the mature capsid have been proposed and may not be mutually exclusive. The most likely process suggests an initial budding of the capsid through the inner nuclear membrane, acquiring an envelope that is then lost as it fuses with the outer nuclear membrane, releasing the naked capsid into the cytoplasm. The association of the capsid with tegument proteins is thought to occur as the capsid buds into the ER, and release of virus from the cell is due to subsequent egress via the Golgi and exocytic vesicles to the plasma membrane. In addition to exocytosis, many herpesviruses invade adjacent cells by the process of cell–cell fusion. The plasma membrane of an infected cell fuses with that of an adjacent uninfected cell, facilitating the entry of progeny virions that undergo a further replication cycle. This phenomenon can be seen in cell cultures, visible as large areas of multinucleate fused cells (syncytia).

Influenza virus and HIV exit the cell by budding at the plasma membrane. Packaging of HIV genomic RNA molecules involves recognition of specific sequences (known as the packaging or epsilon signal).  In a similar manner, accumulation of influenza virus nucleocapsids (RNA segments associated with multiple copies of the viral NP protein and one  copy each of the PA, PB1, and  PB2 proteins) at the plasma membrane leads  to bud- ding. Small sequences in the untranslated regions of each RNA molecule – unique to each segment – may be responsible for selective packaging; insuring one copy of each segment is packaged during budding. Electron microscopy often reveals a distinct pattern of orga- nization of the genomic segments during virus budding, supporting the idea of selective packaging, but it is likely that random packaging also occurs. This theory is supported by the routine isolation of virions containing more than the normal complement of genome molecules (eight  for influenza A virus)  and  also  by the  high  particle to infectivity ratio (Section K6) found in laboratory preparations of virus, suggesting that many virions fail to package a complete ‘set.’