Virus Replication. Initiation Phase: Attachment, Penetration and Uncoating. The replication cycle of HIV is shown below:

Transcription

1 Last Updated: Tuesday, February 16, Virus Replication The replication cycle of HIV is shown below: Initiation Phase: Attachment, Penetration and Uncoating The virion protein or antireceptor binds to a cell surface receptor. A classic example of this process is the haemaglutinin antireceptor of influenza virus. Another intensively studied antireceptor is the gp120 envelope glycoprotein of HIV: Complex viruses such as pox or herpes may have more than one antireceptor. The expression (or absence) of receptors on the surface of cells largely determines the TROPISM of most viruses, i.e. the type of cell in which they are able to replicate - important factor in pathogenesis. Cellular receptors are largely glycoproteins. The interaction of the receptor and antireceptor is (1 of 18)14/05/ :55:48

2 temperature and energy independent. In some cases, attachment leads to irreversible changes in the structure of the virion. The influenza haemaglutinin protein bind to glycoproteins carrying neuraminic (sialic) acid. Attachment is in most cases a reversible process - if penetration does not ensue, the virus can elute from the cell surface. Influenza has a neuraminidase on its surface and can detach from the cell by hydrolysing neuraminic acid from the glycoprotein. This is initially synthesized as a precursor, gp160. A cellular proteases cleave this into gp120 and gp41. gp41 and 120 remain associated as a heterodimer and it is this association which retains the 120 moiety with the virus, since gp41 has the transmembrane domain. The receptor for HIV is CD4 antigen. However, additional structures are also necessary, since transfection of mouse cells with CD4 does not render them permissive for HIV infection. These second receptor components have recently been identified as members of the beta-chemokine receptor family. These receptors normally bind chemokines, small modulators of immune cell function. Examples include CXCR4 and CCR5. The CD4 molecule has 4 regions which resemble the variable regions of antibody molecules. It is a member of the immunoglobulin superfamily of molecules. The binding site for gp120 has been mapped to the first variable region of the protein from amino acids Additional amino acids of the second variable domain may also contribute to binding. Crystal structure of this protein has now been solved. Sequences important for binding CD4 have also been mapped on gp120. A region in the C-terminal half from amino acids associates with it. A deletion of 12 amino acids in this region or site substitutions abolish binding. After attachment, penetration, an energy dependent step, occurs quickly. There are several ways in (2 of 18)14/05/ :55:48

3 which this occurs: 1. Endocytosis of the entire particle resulting in accumulation of virus particles inside a cytoplasmic vesicle. This is said to be obligatory for unenveloped viruses such as polio. Some enveloped viruses, e.g. orthomyxoviruses, also use this method. It is a ph-dependent process 2. Fusion of the cellular membrane with the virion envelope and direct release of the capsid into the cytoplasm examples include paramyxo and herpes viruses as well as HIV. This is phindependent. 3. Rarely, translocation of the virus particle directly into the cytoplasm The precise biophysical details of the fusion process remain unknown. The ph-independent method is used by HIV and Sendai virus (paramyxovirus) and involves direct fusion of the viral envelope with the cell membrane and subsequent release of the capsid into the cytoplasm. The envelope glycoprotein in many viruses including HIV exists as a heterodimer with the extra cellular component (gp120) interacting with the receptor and the transmembrane component (gp41) inducing fusion with the host membrane. The transmembrane protein of enveloped viruses generally contains a stretch of hydrophobic amino acids at the N-terminus implicated in membrane fusion. Anything which disrupts the hydrophobicity disrupts fusion. This process does not result in the formation of an intracellular membrane bound particle, the capsid is released directly into the cytoplasm. In the case of HIV-1 following gp120 binding to CD4 a conformational change facilitates the interaction of gp120 with the chemokine receptor, exposes the fusogenic peptide on gp41 and allows it to insert into the cell membrane... Evidence that viruses infect cells by ph-independent mechanisms can be obtained in several different ways, e.g: EM evidence shows direct fusion with the plasma membrane virus particles remain infectious in alkaline media The ph-dependent process involves endocytosis of a virus. Orthomyxoviruses, such as influenza, enter cells in this way. The receptor bound virus is internalized into the cell by endocytosis. Endocytosed (3 of 18)14/05/ :55:48

4 vesicles form bodies called endosomes. These have an acidic ph which induces a conformational change in the envelope protein resulting in the exposure of a hydrophobic fusion domain. This facilitates fusion of viral and endosomal membranes and release of the capsid particle into the cell. The antireceptor for influenza virus is haemaglutinin. The structure of this protein in several forms has been determined by X ray crystallography. Following binding to the cell surface it is cleaved by a cell associated protease to form a heterodimer of HA1 and HA2. These associate in a trimeric complex.the antireceptor is anchored to the viral membrane by HA2. HA2 has a hydrophobic stretch which is inserted into the viral membrane.. The HA1 chain forms a pocket at the top of this structure which accommodates neuraminic acid of a glycoprotein receptor (ubiquitous). (4 of 18)14/05/ :55:48

5 Cleavage results in a large conformational change which exposes a fusion like domain at the new N- terminus of HA2, strongly conserved, hydrophobic. The virus may be vesicle bound at this stage. From the picture above you can see that the viral and cell membranes are some distance apart (100Ä) after cleavage. The fusion domain is also inaccessible in the interior of HA2. Additional conformational changes induced by ph 5-6 in the endosome bring the fusion domain out of the interior of HA. The two membranes are now linked. HA2 sequences then melt into the lipid bilayer of the cell drawing the viral membrane closer. HA2 known to induce lipid mixing of different bilayers, but mixing of cell contents cannot take place until membrane fusion occurs. A number of chemical agents can help identify which method of uptake is being employed: Lysosomotropic agents such as ammonium chloride, chloroquine (weak bases) can be used to distinguish between ph-dependent penetration and ph-independent penetration because they inhibit the former and not the latter. They are thought to act by acting as weak buffers in endosomes and preventing acidification. Carboxylic ionophores such as monensin and nigericin allow protons to equilibrate between the endosomal and cytoplasm compartments thus preventing acidification DCCD (dicyclohexylcarbodiimide) inhibits the ATPases which pump protons into the vesicle. Entry of unenveloped viruses The uptake of unenveloped viruses usually involves endocytosis into an endosome, i.e. to be phdependent and sensitive to ionophores and lysosomotropic agents. An example is the poliovirus which belongs to the picornavirus family. This is an icosahedral T=3 virus: (5 of 18)14/05/ :55:48

6 The capsid consists of 4 polypeptides, VP1, 2, 3 and 4. VP4 is buried in the capsid and associated with the viral RNA. VP1, 2 and 3 form the external capsid. Five VP1 protein subunits at the 5 fold axis of symmetry form a canyon in the capsid which is the recognition site for the receptor. The polio receptor also belongs to IgG superfamily. It has an amino terminal Ig-like domain which binds to the virus, a transmembrane domain and a cytoplasmic domain (which is dispensible for function. There are several thousand copies of the receptor present on a cell. After binding to the receptor, the virus is endocytosed into an endosome. At a low ph, poliovirus becomes more lipophilic and can associate with the endosome membrane possibly forming a pore. Presumably the ph shift results in exposure of hydrophobic domains in the capsid proteins. During this process VP4 is lost from the particle; VP2 may also be lost. This gives the particle increased flexibility. The genomic RNA is ejected through an endosomal pore into the cytoplasm. (6 of 18)14/05/ :55:48

7 It is possible that poliovirus can also enter the cytoplasm directly in a ph-independent process. Uncoating. This is the general term applied to events after penetration which allow the virus to express its genome. It is poorly understood. In reoviruses, the capsid only ever partially disintegrates and replication takes place in a structured particle. In poxviruses, host factors induce the disruption of the virus. The release of DNA from the core depends upon viral factors made after entry. Orthomyxo, paramyxo and picornavirus all lose the protective envelope or capsid upon entry into the cytoplasm. In the influenza virus an envelope viral protein called M2 may allow endosomal protons into the virion particle resulting in its partial dissolution and permitting replication In herpesviruses, adenoviruses and papovaviruses, the capsid is eventually routed along the cytoskeleton to nuclear envelope. Replication. I have already discussed the diversity of viral genomes and general replication strategies. They will discussed again in more detail in the third year Virology course 335. I will discuss 2 viruses here, HIV a retrovirus with an RNA genome in which the nucleus plays an important role and pox virus, a DNA virus which replicates in the cytoplasm. HIV: Retroviruses first make a double stranded DNA copy of the RNA genome in a process known as reverse transcription. This is thought to take place in a structured remnant of the capsid in the cytoplasm. Little is known about how this event is controlled. It does seem that a cellular protein called cyclophilin is important. This protein interacts with assembling virion particles and is found bound to the gag protein in the particle. Upon entry into the cell the protein apparently helps the dissolution of capsid facilitating reverse transcription.the enzyme concerned is reverse transcriptase. It is both an RNA and DNA directed DNA polymerase. It also has an associated RNAse activity. The process is illustrated below. At this stage it is sufficient for you to know that reverse transcription is primed by a cellular trna towards the 5' end of the genome at the primer binding site. A combination of strand jumps by the newly synthesized DNA in combination with RNA degradation results in the synthesis of a double stranded DNA variant of the genome. If anyone is particularly interested more details are shown below (7 of 18)14/05/ :55:48

8 The proviral DNA is transported to the nucleus and is integrated into the cellular DNA. This can only happen in a dividing cell. HIV is not thought to show any obvious preferences in the sites of integration. The viral enzyme catalyzing this process is called integrase The process is not unlike DNA cutting by (8 of 18)14/05/ :55:48

9 restriction enzymes.both the cellular DNA and the viral DNA are cut by integrase to create sticky cohesive ends. New phosphodiester bonds are formed and the viral DNA has been integrated Organization of the HIV genome: (9 of 18)14/05/ :55:48

10 ~9.5 kilobases long with 3 large open reading frames, gag, pol and env, common to all retroviruses. They encode the structural, enzymic and envelope antireceptor proteins, there are also several smaller reading frames. Four are accessory in the sense that they can be dispensed with in culture: vif - infectivity? vpr - transcription? vpu - env chaperone? nef - indirect effect on transcription? Tat and rev are essential viral proteins involved in the control of gene expression. The gag protein made from an unspliced genomic message. The pol gene overlaps the gag gene by 241 nucleotides. The reading frame of pol is -1 with respect to gag. In about 10% of cases a gag-pol fusion protein is expressed because of a -1 ribosomal frameshifting event, pol also contains a protease activity, integrase activity and reverse transcriptase. gp120 (env) is made from a singly spliced message. The tat and rev genes both have 2 exons and are made from doubly spliced messages. The LTR functions as a highly compressed promoter. Following integration the virus behaves as a resident cellular gene: (10 of 18)14/05/ :55:48

11 Transcription is a complicated process involving the interplay regulatory sequences in the LTR and proteins made by the cell and the virus. RNA polymerase II acts at a promoter in concert with accessory transcription factors (TFs). The accessory transcription factors bind specifically to sequences flanking the promoter and in some cases to RNA polymerase itself. A cell may have hundreds of TFs. Some sites in the LTR function as binding sites for general transcription factors such as the TATA binding factor (TFIID) whilst others bind tissue specific transcription factors e.g. NFkB. NFkB is a particularly important activator in T cells. The presence of many TF binding sites in the viral LTR explains why it is activated in many different cells by many different stimuli. Cellular transcription factors include EBP-1, NFkB and SP1, CTF/NF1, LBP- 1. LTR function simply requires a threshold level of transcription. Significant levels of HIV gene expression are only seen in the presence of tat protein. Tat increases or transactivates mrna production up to a 100X. Tat function requires an RNA sequence known as TAR which is present at the immediate 5' end of all mrnas: (11 of 18)14/05/ :55:48

12 Tar is thought to form a stem-loop structure. Experiments show that tat protein binds directly to TAR RNA.. Evidently the HIV promoter does not assemble a particularly efficient RNA polymerase complex. The initiation rate is slow and elongation efficiency is poor. It is thought that tat binding to TAR recruits other elements to the transcriptional complex which enhance processivity: This RNA-dependent enhancer activity of tat is unique in eukaryotic gene expression. (12 of 18)14/05/ :55:48

13 Initially in an HIV infection small amounts of tat and rev mrna accumulate due to the low endogenous activity of the promoter in the absence of tat. This allows small amounts of tat to be made which then promotes more transcription and more tat in a positive feedback. Over a period of hours although transcription rates remain high the RNA made shifts from small double spliced messages to a single spliced message which encodes env and eventually to an unspliced genomic message which encodes gag and pol. Rev is responsible for this shift in the transcriptional pattern. Again rev binds to the HIV RNA and permits the export of intron containing RNA, a process not normally allowed in the cell. Viral proteins are then made in the cytoplasm by the cellular protein synthesis machinery. Pox Viruses: Large viruses in every sense. Just visible by light microscopy. Genome size of kb, ds DNA but no free ends, connected by a terminal loop. All pox genomes have inverted terminal repeat sequences (ITRs) at the ends of the genome in an opposite orientation. ITR length is variable. Contains in excess of 200 genes. At least 10 genes involved in genome replication. Particles contain upwards of 100 proteins. Most of the essential genes are located in the central part of the genome, while non-essential genes are located at the ends. (13 of 18)14/05/ :55:48

14 Replication occurs in the cytoplasm - the virus is sufficiently complex to have acquired all the functions necessary for genome replication. There is some contribution from the cell but it is not clear what this is - poxvirus gene expression and genome replication occur in enucleated cells, but maturation is blocked. Gene expression is carried out by viral enzymes associated with the core and is divided into 2 phases: Early genes: ~50% genome, expressed before genome replication. These include growth factors, immune defense molecules, enzymes including those involved in DNA replication. These early promoters are AT rich. The RNA polymerase is eukaryotic in character. RNAs are capped and poyadenylated. Uncoating leads to synthesis of DNA genome concatemers. Intermediate genes are expressed giving rise to late transcription factors. Intermediate gene expression may not be possible in virion RNA because of steric inaccessibility to the RNA polymerase or the presence of nucleoid proteins. Late genes are only expressed after genome replication; late promoters are dependent on DNA replication for activity. Virion structural proteins are then made. The concatemeric DNA structures are (14 of 18)14/05/ :55:48

15 resolved in an uncertain fashion and virion particles formed. Intracellular particles carry a Golgi derived envelope. Upon budding they acquire a second envelope from the cell plasma membrane. Assembly, maturation and release. Some viruses make morphogenetic factors which are not structurally part of the virus but whose presence is required for normal assembly. These are sometimes called molecular chaperones. Cellular chaperones may also take part in this process. Other viruses self-assemble. It has been known for more than 40 years that a mixture of TMV proteins and RNA will assemble and make infectious virus in the test tube. This suggests that the TMV structure is a minimum free energy state for its constituent parts. Symmetry is important in viral assembly. It reduces ambiguities in the assembly process and makes it easier. If the coat protein had to form different contacts in different places within the virus structure it would lead to ambiguities in assembly. Filamentous Virus Assembly. In the assembly of TMV the coat protein first forms a disc structure with 17 subunits per ring. This is close to the subunits per turn found in the virus. Close examination of electron micrographs shows that the discs are not actually symmetrical, they have a pronounced polarity: (15 of 18)14/05/ :55:48

16 The disc then interacts with a high affinity binding site on TMV RNA. This converts the disc to a helical "locked washer". Further discs or aggregates of coat protein add to this structure, and also switch to the locked washer. RNA is entrapped in the middle of the disc. Studies show that disc binding is initiated at a unique internal site and that subsequent growth occurs in both directions but at very unequal rates. The major direction of assembly is 3'-5'. This might be expected since the initial nucleation site is close to the 3' end of the virus. RNA is drawn into the assembling structure in a travelling loop. Growth in the 5' direction is fast because a disc can add straight to the protein filament and RNA is drawn up through it. Growth in the 3' direction is slower because the RNA has to threaded through the disc before it can add to the structure. It seems probable that many helical capsids and nucleocapsids will form in a fashion similar to this. Poliovirus forms a procapsid in the cytoplasm. This procapsid nucleic acid which must have specific signals which ensure that only the genomic strand is encapsidated associate with this nascent particle: The nascent polyprotein undergoes several cleavage steps to give the "Monomer-protomer". Five protomers associate to form a pentamer. Twelve pentamers associate to form the icosahedral capsid. RNA associates with the pentamers. This assembly process is self-driven and concentration dependent. Monomers are more stable as pentamers and pentamers are more stable as icosahedral structures. Assembly occurs at lower protein concentrations in the presence of cell extracts than in their absence. This suggests that cellular factors help in this process. RNA association with the pentamer may facilitate icosahedral formation.viruses which are not enveloped like polio usually depend upon disintegration of the cell for their release. The cell often disintegrates because viral replication has prevented normal house keeping functions. (16 of 18)14/05/ :55:48

17 Budding Some viruses, e.g. herpesviruses, assemble in the nucleus. They acquire an envelope as they pass through the inner nuclear membrane. They then accumulate, between the inner and outer lamella of the nucleus. This is topologically equivalent to the cisternae of the cytoplasmic reticulum. From here they pass in a vesicle to the cell surface. These viruses are protected from the cytoplasm. This budding process is invariably cytolytic. Other enveloped viruses acquire their lipid bilayer from the plasma membrane. Capsid assembly can take place in the cytoplasm or in the case of HIV at the plasma membrane. The HIV gag fusion protein p55 is inserted into the lipid membrane by because of a myristyl group at the N-terminus. A patch of gag proteins form associated with the membrane either because of protein-protein interactions or because of association with the viral RNA. The approximately spherical virus may form because of a curvature induced by the gag protein association or because of the helical nature of the RNA. The RNA becomes extensively associated with the nucleocapsid moiety of gag. (17 of 18)14/05/ :55:48

18 The nascent virion then buds through the membrane. The fact that 5% of gag is in the form of a gag-pol fusion which contains reverse transcriptase, integrase and protease ensures that proteins required for infection are present in the virion particle. The relatively high concentration of protease results in its activation and the cleavage of the gag and pol proteins and the eventual formation of the mature and infectious virus.. Other macromolecular constituents of the cytoplasm are also incorporated into the virion unspecifically. The reverse transcriptase primer may be specifically associated with either the viral RNA or the gag protein. This process does not necessarily result in cell lysis and death. Reference: Cann A.J: Principles of Molecular Virology. Academic Press, 2nd Edition, 1997 Chapter 4. Search for more information on this topic. Return to Course 224 HomePage Dr Shaun Heaphy. (18 of 18)14/05/ :55:48