Kate Bishop group

Infection and replication of retroviruses

Retroviruses cause severe diseases, including immunodeficiency and cancer. The human immunodeficiency virus (HIV) is the most widely known retrovirus due to its impact on human health. The latest figures report that 33 million people globally are living with HIV/AIDS.

Innovative therapeutics for retroviral diseases will hopefully arise from a better understanding of how retroviruses reproduce in the cell, how they interact with host cell factors and how they subvert the host innate and adaptive immune systems. The early stages of the retroviral life cycle are particularly attractive therapeutic targets, with several anti-retroviral drugs and cellular anti-viral factors inhibiting these steps. However, numerous events that occur during these stages are still poorly understood. The three main projects in our laboratory aim to characterise the molecular events that occur once a retrovirus has entered a cell in order to fully understand retroviral replication and provide potential ways in which to manipulate these processes for the benfit of human health.

The life cycle of a retrovirus

The life cycle of a retrovirus

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The retroviral life cycle is arbitrarily divided into two phases, early and late. The stages in each phase are shown above. Interactions between viral and host cell factors occur at every stage of the viral life cycle, although many are still poorly understood. Identifying and understanding these interactions are key to developing new treatments to combat retroviral diseases. The steps inhibited by three retroviral restriction factors TRIM5alpha, APOBEC3G and Fv1 are also indicated. (RTC, reverse transcription complex; PIC, pre-integration complex)

The first project in the laboratory focuses on the p12 protein from the prototype retrovirus, murine leukaemia virus (MLV). All retroviral genomes contain a gag gene that codes for the Gag polyprotein. Gag is cleaved upon viral maturation to release individual proteins, including matrix, capsid and nucleocapsid, providing the structural components of the virion. In murine leukaemia virus (MLV), Gag cleavage releases an additional protein, named p12, required for both early and late stages of the viral life cycle. The role of p12 during early events is unknown, and it is the only MLV protein without a function-associated name. Viruses carrying mutations in p12 are able to reverse transcribe their genomes but cannot integrate this nascent DNA.

Gag proteins and p12

Gag proteins and p12

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Retroviral Gag polyproteins are processed into at least three proteins, matrix (MA), capsid (CA) and nucleocapsid (NC), that form the structure of the virion. Most retroviruses also contain additional Gag cleavage products, for example, the p12 protein of MLV and the p6 protein of HIV-1. The full amino acid sequence of Moloney-MLV p12 is shown, with the blocks of residues that are changed to alanines in our panel of p12 mutants highlighted in colour. Viruses carrying these mutant p12 proteins cannot complete the early stages of replication.

Using mutagenesis studies, we have mapped two functional domains in p12 that act in concert and can behave in a dominant negative manner. We have also purified p12 and shown that it does not self associate in vitro. Based on our data, we have proposed a model for p12 function during the early stages of retroviral replication. By combining virological assays with biochemical/biophysical techniques and microscopy, we aim to build up a picture of how p12 interacts with both viral and cellular factors and define the mechanism of p12 function in various gammaretroviruses. In addition, we are using fluorescent microscopy to visualise p12 localisation during viral replication. We will compare fluorescent and electron microscopy and are developing methods to watch infection in real-time.

Model for p12 function

Model for p12 function

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In addition to the late (L)-domain, we propose that p12 carries two early domains in the N-terminus (Early-A, E-A, green box) and the C-terminus (Early-B, E-B, purple box). (A) Infection of a cell with a gammaretrovirus containing wild type p12 leads to successful integration of viral cDNA into the host chromatin. (B) Alterations to the N-terminal domain of p12, E-A, affect the stability of the viral core and abort infection very early in the replication pathway, sometimes inhibiting reverse transcription. The virus is therefore unable to abrogate restriction factors. (C) Alterations to the C-terminal domain of p12, E-B, do not affect the very early stages of infection and p12 is present in the preintegration complex (PIC) by virtue of interactions at the N-terminus. However, p12 is unable to tether the PIC to host chromatin and the viral cDNA cannot integrate successfully into the DNA of the host.

The second project aims to unravel the antiviral mechanism of the newly identified restriction factor SAMHD1. This protein inhibits HIV replication in cells of the myeloid lineage and may help to preserve the latent HIV reservoir in patients. The HIV-2/SIV protein Vpx overcomes SAMHD1 restriction. In collaboration with other groups at NIMR, we are currently correlating SAMHD1 expression levels and enzymatic activity with cellular dNTP levels and viral infectivity using a panel of SAMHD1 variants, including mutations linked to Aicardi-goutières Syndrome, together with reverse transcriptase and Vpx mutants. We are also assessing alternative methods of modulating dNTPs levels in the cell and characterising the effects of such modulation on HIV replication, immune activation and cellular metabolism.

Model for SAMHD1 function

Model for SAMHD1 function

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(A), Dimeric inactive SAMHD1 (blue) binds dGTP at the dimer interface. Activated SAMHD1 (red) catalyses the cleavage of dNTPs into the composite deoxynucleoside and inorganic triphosphate. SAMHD1 activity in myeloid cells suppresses the deoxynucleotide pool, inhibiting reverse transcriptase and blocking infection by HIV-1. (B) In the presence of Vpx, SAMHD1 is recruited by the DDB–CUL4–DCAF1 E3 ubiquitin ligase complex and targeted to the proteasome for degradation. Control on the dNTP pool is released and sufficient dNTPs are available for reverse transcription to be completed, allowing infection by HIV-2 and SIVs that encode Vpx. (C) AGS mutations in the allosteric binding site of SAMHD1 prevent dGTP binding or allosteric activation rendering the protein inactive. As a result, deoxynucleotide levels rise and aberrant DNA products arising from reverse transcription of endogenous retroviruses accumulate within the cytoplasm, triggering the inappropriate production of interferon observed in AGS.

The latest project in the laboratory is to establish the relationship between uncoating, reverse transcription, cytoplasmic trafficking and nuclear entry of HIV, and to identify host factors that are involved in these processes. Using viral mutants and chemical inhibitors to alter specific processses known to be required for infection, we can measure the kinetics of various replication steps, comparing cell-based systems with in vitro assays to determine what events trigger others and what contribution the cell makes to the timing and order of replication steps. In the course of these studies we additionally hope to identify viral variants and/or procedures that are ameniable to biophysical examination, for example isolating preintegration complexes that could be studied using cryo-electron microscopy.

Selected publications

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