Kate Bishop: Projects

The function of the MLV p12 protein

The laboratory has been studying the function of 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. We are characterising the role of p12 during early events. Viruses carrying mutations in p12 are able to reverse transcribe their genomes but cannot integrate this nascent DNA.

 

Gag proteins and p12

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. (Click to view larger image)

 

Using mutagenesis studies combined with microscopy and biochemical experiments, we mapped two functional domains in p12. We showed that the N-terminal motif of p12 binds directly to the capsid protein and that mutations in this motif affect the structure and stability of the viral capsid shell. Additionally, we have demonstrated that p12 is involved in directing the viral pre-integration complex to chromatin ready for integration. We are continuing to use p12 to unravel the key processes of capsid shell formation and break down (uncoating) and chromatin tethering, all of which are required for all retroviral infections.

Model for p12 function

(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. (Click to view larger image)

 

The function of the HIV capsid protein

The relationship between uncoating (breakdown of the viral core), reverse transcription, trafficking the core to the nucleus and nuclear entry of HIV remain unclear. However, recent evidence suggests that the capsid protein (CA) is central to all early, post-entry replication steps, including nuclear events. The discovery that p12 is essential for MLV integration and interacts with CA supports this notion (see above). The mechanism of uncoating, when and where in the cell it occurs, and which, if any, host proteins contribute to the process are controversial. However, most now agree that perturbing uncoating is detrimental to infectivity and that uncoating is a regulated process. Indeed, uncoating is considered a potential therapeutic target.

Using viral mutants and chemical inhibitors to alter reverse transcription we measured the kinetics of uncoating and determined that HIV-1 uncoating is triggered by a specific step of reverse transcription. This suggests possible mechanisms that drive the uncoating processthat we are continuing to investigate. We are also capitalising on expertise in cryo-electron microscopy at The Crick to develop a novel system to visualise uncoating intermediates that have proved elusive to this point. Knowing the amount and arrangement of CA in the PIC will help elucidate the function of CA during integration. To further these studies we have also characterised a panel of HIV-1 mutants with altered CA shell stabilities. Using these, together with a new fluorescent microscopy technique we are exploring the localisation of CA and the interactions between CA and cellular factors.

The function of the HIV accessory proteins, Vpx and Vpr

The HIV-2 protein Vpx enhances replication of both HIV-1 and HIV-2 in cells of the myeloid lineage and resting T cells. In 2011, the target for Vpx was identified as the cellular protein SAMHD1 (sterile alpha motif and an histidine-aspartate domain-containing protein 1). Vpx recruits SAMHD1 to the DDB1/CUL4A/ROC1 E3 ubiquitin ligase complex through interaction with the substrate-adaptor protein DCAF1 resulting in proteasomal degradation of SAMHD1. Mutations in SAMHD1 in humans lead to Aicardi Goutières syndrome (AGS), which mimics congenital viral infection. Ian Taylor's group at the Crick demonstrated that SAMHD1 is a GTP/dGTP-activated deoxynucleotide triphosphohydrolase that degrades dNTPs to constituent nucleoside and inorganic triphosphate. This led to the hypothesis that SAMHD1 inhibited HIV-1 replication by reducing cellular dNTP pools in differentiated cells below the threshold required for reverse transcription, although alternative mechanisms have also been proposed. In collaboration with the Taylor group, we have been pursuing how SAMHD1 inhibits HIV-1 replication and investigating how Vpx recruits SAMHD1 and targets it for degradation.

Model for SAMHD1 function

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. (Click to view larger image)

 

Vpr is a paralogue of Vpx with an unknown function in HIV-1. Expression of Vpr induces cell-cycle arrest, and, as Vpr binds the substrate-adaptor protein DCAF1, it seems likely this is due to Vpr targeting a cellular protein for degradation. Several Vpr targets have been proposed, although none appear responsible for the cell cycle block, and there is no mechanism for how they inhibit HIV-1 replication. In addition, Vpr proteins from some SIV strains can induce degradation of SAMHD1. Continuing our work on SAMHD1 and Vpx, we are now focusing on deducing the function of Vpr. We are using cutting edge techniques to identify novel cellular target(s) for Vpr and reveal their antiviral function(s). Furthermore, we are investigating the specificity and evolutionary conservation of several recently reported Vpr interactions using our established cell-based degron assay, in vitro assays and structural studies in collaboration with Ian Taylor.

Kate Bishop

kate.bishop@crick.ac.uk
+44 (0)20 379 62431

  • Qualifications and history
  • 1998-2001: PhD in Virology, MRC-NIMR affiliated to UCL, London, UK
  • 2002-2004: Post doctoral researcher, King's College London, UK
  • 2004-2008: Royal Society Dorothy Hodgkin Fellow, King's College London, UK
  • 2008-2014: Wellcome Trust Career Development Fellow, MRC-NIMR, London, UK
  • Since 2008: Group Leader, Medical Research Council National Institute for Medical Research, London, UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK