Chapter 4: ReplicationSlide 1/25Replication Part 2 Part 1Chapter 4: ReplicationSlide 2/25Cell TropismIn many if not most cases, the expression (or absence) of receptors on the surface of cells largely determines the tropism of a virus, i.e. the type of host cell in which it is able to replicate.In some cases, intracellular blocks at later stages of replication are responsible for determining the range of cell types in which a virus can carry out a productive infection, but this is not common.Therefore, this initial stage of replication however, the internalization step is more selective, giving rise to a degree of cell selection.Chapter 4: ReplicationSlide 4/25Multiple Receptors (2)The primary receptor for HIV is the helper T cell differentiation antigen, CD4. Transfection of human cells which do not express CD4 with recombinant CD4-expression constructs makes them permissive for HIV infection. However, transfection of rodent cells with human CD4-expression vectors does not permit productive HIV infection. If HIV provirus DNA is inserted into rodent cells by transfection, virus is produced, so there is no intracellular block to infection. Therefore, there must be one or more accessory factors in addition to CD4 required to form a functional HIV receptor. These are a family of proteins known as b b-chemokine -chemokine receptorsreceptors. Several members of this family have been shown to play a role in the entry of HIV into cells, & their distribution may be the primary control for the tropism of HIV for different cell types (lymphocytes, macrophages, etc). Chapter 4: ReplicationSlide 5/25Penetration Penetration of the target cell normally occurs a very short time after attachment of the virus to its receptor in the cell membrane. Unlike attachment, cell penetration is generally an energy-dependent process, i.e. the cell must be metabolically active for this to occur. Three main mechanisms are involved:Chapter 4: ReplicationSlide 6/25Translocation1) Translocation of the entire virus particle across the cytoplasmic membrane of the cell.This process is relatively rare among viruses & is poorly understood.It is mediated by proteins in the virus capsid & specific membrane receptors.Chapter 4: ReplicationSlide 7/25Endocytosis2) Endocytosis of the virus into intracellular vacuoles is probably the most common mechanism of virus entry into cells. Fusion does not require any specific virus proteins (other than those already utilized for receptor binding) but relies on the normal formation & internalization of coated pits at the cell membrane. Receptor-mediated endocytosis is an efficient process for taking up & concentrating extracellular macromolecules.Chapter 4: ReplicationSlide 8/25Fusion3) Fusion of the virus envelope (enveloped viruses only) with the cell membrane, either directly at the cell surface or in a cytoplasmic vesicle. Fusion requires the presence of a specific fusion protein in the virus envelope, e.g. influenza haemagglutinin or retrovirus transmembrane (TM) glycoproteins, which promotes joining of the cellular & virus membranes which results in the nucleocapsid being deposited directly in the cytoplasm. There are two types of virus- driven membrane fusion: pH- dependent & pH-independent.Chapter 4: ReplicationSlide 9/25UncoatingUncoating is a general term for the events which occur after penetration, in which the virus capsid is completely or partially removed & the virus genome exposed, usually in the form of a nucleoprotein complex. Uncoating is one of the stages of virus replication that has been least studied & is relatively poorly understood. The product of uncoating depends on the structure of the virus nucleocapsid. In some cases, this might be relatively simple (e.g. picornaviruses have a small basic protein of approximately 23 amino acids (VPg) covalently attached to the 5 end of the vRNA genome), or highly complex (e.g. retrovirus cores are highly ordered nucleoprotein complexes which contain, in addition to the diploid RNA genome, the reverse transcriptase complex). The structure & chemistry of the nucleocapsid determines the subsequent steps in replication.Chapter 4: ReplicationSlide 10/25Genome Replication & Gene ExpressionThe replication strategy of any virus depends on the nature of its genetic material.All viruses can be divided into seven groups - such a scheme was first proposed by David Baltimore in 1971.Originally, this classification included only six groups, but it has since been extended to include the scheme of genome replication used by the hepadnaviruses & caulimoviruses.For viruses with RNA genomes in particular, genome replication & the expression of genetic information are inextricably linked, so both of these criteria are taken into account in this scheme.Chapter 4: ReplicationSlide 11/25Class I: Double-stranded DNAThis class can be subdivided into two further groups:A)R