Virus Replication

Human immunodeficiency virus (HIV-1) will be discussed in detail here as an example of virus replication. In some cases, comparisons between HIV-1 replication and that of other viruses will be made, either because other viruses are better understood or to illustrate variations in these processes.

Infection of a cell may be:

• Productive: the cells are permissive for viral replication and virion progeny are released
• Abortive: the cells are non permissive for a viral function and virion particles not produced.
• Restrictive: the cell is transiently permissive and a few virus are produced. Viral production then ceases but the genome persists. Examples include Epstein Barr and herpes simplex. May still have serious consequences for the host.

The replication cycle of HIV is shown:

All viruses have to penetrate, replicate and exit a cell. The details of how they do this obviously vary. The Baltimore classification is a useful way to order this information. The replication strategy of the virus depends on the nature of its genome. Viruses can be classified into seven (arbitrary) groups:

I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc)

II: Single-stranded (+)sense DNA (Parvoviruses)

III: Double-stranded RNA (Reoviruses; Birnaviruses)

IV: Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc)

V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc)

VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle (Retroviruses)

VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses)

Class I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses; Papovaviruses):

a) Replication is exclusively nuclear (e.g. Adenoviruses, Papovaviruses, Herpesviruses).
Transcription of virus genome occurs in successive rounds:

Immediate-Early      }         Dependent on virus encoded
Early                }         trans-acting factors.
Late

Class II: Single-stranded (+)sense DNA (Parvoviruses):

Class III: Double-stranded RNA (Reoviruses):

Class IV: Single-stranded (+)sense RNA (Picornaviruses; Caliciviruses; Togaviruses; Flaviviruses; Coronaviruses):

a) Polycistronic mRNA e.g. Picornaviruses; Hepatitis A. Genome RNA = mRNA. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins.

b) Complex Transcription e.g. Togaviruses. Two or more rounds of translation are necessary to produce the genomic RNA.

Class V: Single-stranded (-)sense RNA (Orthomyxoviruses; Paramyxoviruses; Rhabdoviruses; Filoviruses; Bunyaviruses):

a) Segmented e.g. Orthomyxoviruses. First step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for genome replication.
b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mRNAs are produced.

Class VI: Single-stranded (+)sense RNA with DNA intermediate (Retroviruses):

Class VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses):

To successfully replicate its genome, the virus must present mRNA to the cell which can be recognized and translated into virus-encoded proteins. This can be done in a number of ways, e.g:

• d/s DNA viruses release their genome into the nucleus and rely on the cellular machinery for transcription.
• (+)sense RNA viruses have genomes which serve directly as mRNA (except retroviruses!)
• (-)sense RNA viruses must carry with them virus-coded enzymes for RNA-dependent RNA replication, since uninfected cells do not posses these enzymes.

Virus replication can be divided into (arbitrary) phases:

Initiation:

• attachment
• penetration
• uncoating

Replication:

• genome synthesis
• RNA production
• protein synthesis

• ensure replication of the genome
• package the genome into virus particles
• alter the metabolism of the infected cell so that viruses are produced.

In a few instances, e.g. parvoviruses, it is cellular enzymes which replicate the viral genome and these are just assisted by viral proteins. But in most cases viral proteins are responsible for genome replication, albeit with the assistance of cellular proteins. It is viral proteins that are responsible for the third and final stage, i.e: Release:

• assembly
• maturation
• exit from cell

Initiation Phase:

Attachment, Penetration and Uncoating

Cellular receptors are largely glycoproteins. The interaction of the receptor and antireceptor requires counter ions to reduce electrostatic repulsions and is 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. The virus also has a neuraminidase on its surface and can detach from the cell by hydrolysing neuraminic acid from the glycoprotein.

The antireceptor of HIV is the gp120 envelope 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 proteolysis event is essential for virus infectivity.

The receptor for HIV is CD4 antigen, which binds gp120 with an affinity constant in the nanomolar range. Binding has been demonstrated by coprecipitation experiments using antibodies to either protein.

Definitive proof that CD4 is the HIV receptor was obtained by Maddon and Weiss. They showed that CD4 negative human cells (e.g. HeLa cells) cannot be infected with HIV. Expression of CD4 in these cells following transfection with a cloned CD4 gene made them permissive. 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 family, in particular CXCR-4 and CCR-5.

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 using blocking antibodies and site-directed mutagenesis. It maps to the first variable region of the protein from amino acids 16-84. 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 397-439 associates with it. A deletion of 12 amino acids in this region or site substitutions abolish binding.

More detailed understanding of the interactions of these two proteins is now dependent on knowing the tertiary structure of gp120. This is particularly important because it is a potential therapeutic site.

After attachment, penetration, an energy dependent step, occurs quickly. There are several ways in 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 (but see below). 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 pH-independent.

3. Rarely, translocation of the virus particle directly into the cytoplasm

The precise biophysical details of the fusion process remain unknown. Some viruses may enter the cell in more than one way. With other viruses the evidence for an entry mechanism is not always absolutely conclusive.

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. (Confusingly, it seems that other retroviruses may be internalized by a pH-dependent process.) 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. HIV is no exception and mutations in this region abrogate cell fusion. Anything which disrupts the hydrophobicity disrupts fusion. Mutations which enhance hydrophobicity actually enhance the fusogenic potential of viruses. This process does not result in the formation of an intracellular membrane bound particle, the capsid is released directly into the cytoplasm.

Evidence that viruses infect cells by pH-independent mechanisms can be obtained in several different ways:

• EM evidence shows direct fusion with the plasma membrane
• virus particles remain infectious in alkaline media
• In the case of HIV, a truncated CD4 with cytoplasmic domain deleted still functions as a receptor despite not being internalized in response to phorbol esters.

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 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. The virus contains two other membrane proteins, the neuraminidase and M₂ proteins.

Haemaglutinin is made as a precursor protein with 549 amino acid residues. It is glycosylated and stabilized by several disulphide bonds. The antireceptor is anchored to the viral membrane by HA₂. HA₂ has a hydrophobic stretch from 185-211 which is inserted into the viral membrane . The N-terminal sequence forms a trimeric structure of three HA₂ held together by hydrophobic bonds. The HA₁ chain forms a pocket at the top of this structure which accommodates neuraminic acid of a glycoprotein receptor (ubiquitous).

Native haemaglutinin is cleaved by a cell surface protease after amino acid 328 into HA₁ and HA₂ (221 amino acids). The two polypeptide chains remain tightly associated after this cleavage. They are held together by both non covalent and covalent bonds. 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 (100Å) 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. An extended loop sequence changes conformation to a coiled coil and projects the fusion domain into the cell membrane. 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 (dicycohexylcarbodiimide) inhibits the ATPases which pump protons into the vesicle.

Entry of unenveloped viruses

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 belongs to IgG superfamily (as does CD4), it has a MW of 45Kd. 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.

It is possible that poliovirus can also enter the cytoplasm directly in a pH-independent process. It has been demonstrated that the purified viral receptor converts the 160S poliovirus particle into a 135S form lacking VP4. This further dissociates to an 80S complex in which the RNA has been lost. It has also been observed that an "engineered" receptor molecule which lacks the cytoplasmic domain and in its place contains a GPI anchor preventing internalization still confers susceptibility to infection. It is possible that the hydrophobic amino terminus of VP1 is exposed on the surface of the capsid during the transition from a 160S complex to a 135S complex and that the capsid then fuses directly with the cell membrane.

Uncoating.

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.

Orthomyxoviruses, paramyxoviruses and picornaviruses 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. M2 is a polypeptide of 97aa It forms a tetrameric channel forming structure within the viral envelope. Amantadine and rimantidine are anti-influenza drugs which function in part by inhibiting M2. In cells treated with rimantidine the nucleocapsid remains associated with matrix protein and does not go to the nucleus.

In herpesviruses, adenoviruses and polyomaviruses, the capsid is eventually routed along the cytoskeleton to nuclear envelope.

Replication

In retroviruses, the matrix protein probably stays associated with cytoplasmic membrane after entry. Reverse transcription ie copying the RNA genome into a double stranded DNA form is thought to take place in a structured remnant of the capsid in the cytoplasm. 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:

It results in the formation of a double-stranded proviral DNA which is longer than the viral RNA by one copy of U3, R and U5. In reality, the procedure is more complicated than shown. There are probably more than 2 strand jumps because retroviral RNA is often broken or nicked. At every nick of necessity the RT complex has to switch to another RNA strand (remember that there are two present).

The proviral DNA is transported to the nucleus and integrated into the cellular DNA. HIV is not thought to show any obvious preferences in the sites of integration. The viral enzyme catalyzing this process is called integrase.

A linear form of the proviral DNA is integrated. This reaction is unique to retroviruses. Two bases are first removed from the linear DNA at the 3' end to give a 3' recess. A staggered cut 6 nucleotides apart is then made in the genomic DNA and the resulting 5' phosphates are ligated to the 3' OH groups. This leaves a gapped intermediate which is repaired. The reaction can take place in vitro in the absence of ATP. Cleavage always occurs at the same place in the LTR. The reaction does not go to completion in quiescent T cells.

Organization of the HIV genome:

~9.5 kilobases long with 3 large open reading frames, gag , pol and env , common to all retroviruses. 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 slippage. This occurs within a sequence of 6U residues, U UUU UUA and means that 1 base is read twice. The process is called ribosomal frameshifting and occurs in other viral families, pol also contains an aspartate protease activity. This cleaves the gag/pol fusion protein. It further cleaves pol to liberate integrase, reverse transcriptase and free protease.

gp120 (env) is made from a singly spliced message.

The tat and rev genes both have 2 exons, partially overlap each other in different reading frames, made from doubly spliced messages.

The LTR functions as a highly compressed promoter. Following integration the virus behaves as a resident cellular gene:

Transcription is a complicated process involving the interplay of cis-acting regulatory sequences in the LTR and trans-acting proteins made by the cell and the virus.

RNA polymerase II acts at a promoter in concert with accessory transcription factors (TFs). These 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:

Tar is thought to form a stem-loop structure. Experiments show that tat protein binds directly to TAR RNA with a Kd of about 1nM. One molecule of tat binds to RNA. Mutagenesis experiments show that binding is dependent on the presence of a U bulge in the TAR element. TAR is only functional as an RNA element. TAR only functions in a position immediately 3' of the site of transcription initiation. This RNA-dependent enhancer activity of tat is unique in eukaryotic gene expression.

Other experiments show that the HIV promoter does not assemble a particularly efficient RNA polymerase complex. The initiation rate is slow and elongation efficiency (processivity) is poor. It is thought that tat binding to TAR recruits other elements to the transcriptional complex which enhance processivity:

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. The rev protein controls this 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. It down regulates tat and rev RNA in a negative feedback. The rev response involves a cis element of about 250 bases present in env . This for obvious reasons has been called the rev-response element or RRE, again it is only functional as an RNA element. Rev binds directly to the RRE with a Kd of 1nM. The position of the RRE explains why the double spliced messages are not rev sensitive, i.e. because they are spliced out. Rev has been suggested to control either RNA splicing or nuclear-cytoplasmic transport or translation. Recently it has been shown to associate with nucleoporin proteins that are thought to be found in the nuclear pore complex.

Protein translation is another control point during viral replication. Interferon is made in response to viral infection. It switches on more than 30 other genes. 2'5' polyadenylate synthetase makes 2'-5' linked polyadenylates from ATP . These activate a protein called RNAse L which degrades cellular and viral RNAs. Many viruses produce double stranded RNA during replication. These can activate another enzyme known as PKR. PKR is a protein kinase. When it is activated it phosphorylates the alpha factor of protein initiation factor eIF2:

This then sequesters a guanine nucleotide exchange factor which is essential for the initiation of protein synthesis and is present in very low amounts. This results in the cessation of host cell protein synthesis prevents virus replication: Many viruses have evolved strategies to down regulate PKR:

• Reoviruses and various pox viruses make proteins which sequester the activating RNAs
• Pox viruses also makes a "suicide" pseudosubstrate for PKR
• Poliovirus expresses a protease which degrades it
• Influenza induces synthesis of a cellular repressor
• EBV (HHV-4) and adenovirus make RNAs which bind to the enzyme but do not activate it and prevent other RNAs from activating it.

HIV may behave like EBV (HHV-4) and adenovirus. The TAR sequence which is double-stranded binds to PKR but does not activate it. TAR is ideally placed to do this because it is at the 5' end of all HIV mRNAs and PKR is associated with ribosomes. This gives rise to the possibility that preferential translation of HIV mRNA may occur in cells where host protein synthesis has largely been shut off.

Assembly, maturation and release.

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.

In the assembly of TMV the coat protein first forms a disc structure with 17 subunits per ring. This is close to the 16.34 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:

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.

Budding

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.

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 which was discussed at the beginning of this document. The fact that the env glycoprotein has a symmetry related to an icosahedron suggests that it may interact with the matrix protein underlying the lipid bilayer and that its incorporation into the virion particle does not just depend upon the envelope glycoprotein being present in the lipid bilayer. 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.

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