In the three and a half decades since HIV was discovered researchers have gained a strong understanding of the virus; however, some features remain largely uncharacterized. The HIV genome encodes essential virion proteins common to nearly all retroviruses. HIV also encodes four accessory factors that enhance viral infectivity yet are not essential in tissue culture. Vpr is a particularly interesting protein expressed in HIV-1, HIV-2, and simian immunodeficiency viruses (SIV). While Vpr is present in all these viral lineages, its role in the viral life cycle remains a question of debate. Studies of HIV-1 Vpr have suggested it is involved in transcriptional activation, nuclear import, apoptosis, and DNA damage response. These diverse results may be a consequence of Vpr having multiple functions or alternatively, they may represent particular host adaptations of viruses to particular species. Studying viral evolution is important because viral-host interactions drive a rapid, species-specific adaptation, thus expressing SIV proteins in human cells (or vice versa) may give insights into the importance of different activities during an actual infection in their natural hosts. Before starting his own lab at UCLA, former Fred Hutch postdoc Dr. Oliver Fregoso analyzed the role of HIV-1 and 2 Vprs in activating DNA damage repair. His work in the Emerman Lab (Human Biology Division) was recently published in mBio and has helped to both eliminate and validate possible Vpr activities.
Vpr in HIV-1 is known to activate the DNA damage repair pathway, and that this activity is dependent on the DNA damage response protein ATR, yet the direct interactions that stimulate this pathway are unclear. Recent evidence suggested Vpr stimulated DNA damage repair by directly binding and activating the endonuclease SLX4, thus generating lesions at presumed replication forks. In this study Drs. Fregoso and Emerman evaluated Vpr activity on DNA damage and SLX4 binding in a broader range of human and simian viruses. First they found that HIV-1 Vpr and HIV-2 Vpr robustly stimulated DNA damage repair; however, only HIV-1 Vpr co-immunoprecipitated with SLX4. However, the interaction between HIV-1 Vpr and SLX4 appears to be a poorly conserved activity specific to other HIV-1 strains. After testing 18 strains of HIV and also some related SIVs from the HIV-1 and HIV-2 evolutionary branches only 4 Vprs co-immunoprecipitated with SLX4, although all of them could cause a DNA damage response. These results strongly suggested a conserved function for Vpr proteins to activate DNA damage repair pathways, but SLX4 binding is likely not required.
CRISPR-mediated knock-out (KO) of SLX4 allowed researchers to directly test if SLX4 was required to stimulate DNA damage repair pathways after Vpr expression. Two SLX4 KO clones were isolated and SLX4 deletion was sequence verified. Both clones were viable but sensitized to DNA damaging agents. HIV-1 and HIV-2 Vpr proteins were expressed in these clones, and both induced DNA damage repair signaling whether or not SLX4 was present. This made it clear SLX4 was not required to stimulate the response, however, it remains untested if SLX4 binding may serve as a redundant pathway in those few HIV-1 strains.
Finally, researchers demonstrated that more evolutionarily distant SIV Vpr homologs failed to bind SLX4, yet they had not established that SIV Vpr even stimulates DNA damage repair signaling. In spite of unique virus-host interactions, SIV Vpr proteins with little similarity to HIV-1 or HIV-2 Vprs stimulated DNA damage repair pathways when expressed in human cells. This result is important for future work, as it indicates these orthologs can be studied in the same cellular context. The evolutionary approach used in this study may be useful for future analysis of Vpr as Dr. Fregoso explained, “HIV-1 and HIV-2 Vpr have distinct cellular localization patterns. HIV-1 Vpr has been suggested to have a role in nuclear import, but those results have not been tested with different orthologs, which could help determine if the localization matters for this. What is also interesting is that it seems HIV-1, and to an extent SIVcpz, are the outliers – all other Vpr proteins have a pattern similar to HIV-2 Vpr.”
Moreover, this finding brings researchers closer to understanding Vpr functions. “We have known for a long time that Vpr causes cell cycle arrest and a DNA damage response. The work in the mBio paper establishes that this role is conserved throughout primate lentivirus evolution, so there is a likely a selection for this activity. However, what we still don’t know is whether this is a primary role or a by-product of something else that Vpr is doing that is more important.” said Dr. Emerman.
Continued work at Fred Hutch and now UCLA is expanding our knowledge of lentiviruses and their unique biology. A fundamental understanding of how these viral proteins function enables advances in other fields such as biotechnology, and medicine.
Fregoso OI, Emerman M. 2016. Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment. MBio, 13;7(5).
Funding for this research was provided by the National Institutes of Health and Burroughs Wellcome Fund