CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

201 The Activity of HIV-1 Rev/RRE Varies Greatly Between Isolates Patrick E. Jackson 1 ; DenisTebit 1 ; David Rekosh 1 ; Marie-Louise Hammarskjold 1

1 University of Virginia, Charlottesville, VA, US; 2 University of Virginia, Charlottesville, VA, US Background: HIV replication is a highly regulated process, and the binding of the viral Rev protein to the Rev Response Element (RRE) is essential for the nucleocytoplasmic export of mRNAs that retain introns and thus for packaging of genomic RNA. Previous studies have shown that there is variation in Rev and RRE sequence both within and between patients. We previously assessed the activity of the Rev-RRE system in viruses isolated from patients. Variation of Rev/RRE function was observed between patients and within a patient as the infection progressed. In the present study, we sought to examine whether Rev/RRE functional variation could help to explain changes in the relative prevalence of HIV subtypes in populations. Methods: A functional assay was developed, which utilizes an HIV vector system that packages HIV vector RNA into particles in a Rev-dependent fashion. The virus particles formed in this system transduce hygromycin resistance to target cells, allowing Rev/RRE activity to be read-out by counting the number of resistant colonies. Rev and RRE sequences can be tested in this system to measure the function of naturally occurring or artificial Rev-RRE pairs. Eight sequences were selected from the Los Alamos database representing two subtype A, four AG, and two G examples. Four subtype B patient isolates were also included. Results: There was a 24-fold difference in functional activity between the most and least active naturally occurring Rev/RRE combinations (cognate pairs). There was no clustering within withsubtypes. When selected Rev and RRE sequences were tested with NL4-3 RRE and Rev, the absolute activity could not be predicted from the activity of the cognate pair. In a linear regression analysis, there was low correlation of RRE activity (R 2 =0.32) but high correlation of Rev activity (R 2 =0.95) with the functional activity of the original cognate pair. Conclusions: Rev/RRE functional activity varied dramatically between naturally occurring cognate pairs. This functional variation appeared to be primarily driven by differences in Rev. The clinical significance of this finding is still unclear, but our results demonstrate that there are clear differences in the Rev activity of circulating viruses. This may play a role in viral fitness and replication dynamics and be reflective of immune pressure. Identification of the most efficient Rev/RRE combination would also likely be of use in optimizing lentiviral vector systems for gene therapy.

TUESDAY, FEBRUARY 24, 2015 Session P-A3 Poster Session

Poster Hall

2:30 pm– 4:00 pm SAMHD1 202 A Surprising New Function of SAMHD1 as a Pro-Pathogenic Factor in HIV Infection

Gilad Doitsh ; Nicole Galloway; Xin Geng; Isa Monus Arias; ZhiyuanYang;Warner C. Greene The J. David Gladstone Institute, University of California San Francisco, San Francisco, CA, US

Background: Depletion of CD4 T cells and development of chronic inflammation are signature processes in HIV pathogenesis that propel disease progression. Due to endogenous SAMHD1 restriction activity in quiescent lymphoid CD4 T cells, the viral chain elongation phase of reverse transcription is attenuated, giving rise to incomplete cytosolic DNA transcripts. CD4 T-cell death is triggered after sensing of these cytosolic DNA intermediates by interferon gamma Inducible protein 16 ( IFI16 ). Death occurs following caspase-1 activation in inflammasomes and the induction of pyroptosis , a highly inflammatory form of programmed cell death. These findings mechanistically connect CD4 T-cell death and chronic inflammation––the two signature pathogenic processes of active HIV infection. Methods: Human lymphoid aggregated cultures (HLACs) prepared using tonsil and spleen, and lymph node biopsies from consenting HIV-infected volunteers were used. Results: We now show that SAMHD1 restriction activity influences how CD4 T cells die. Degradation of SAMHD1 by Vpx encoded by HIV-2 thwarts abortive infection in resting, non-permissive lymphoid CD4 T cells redirecting the cell death pathway away from caspase-1-mediated pyroptotic pathway (inflammatory) toward caspase-3-mediated apoptotic pathway (noninflammatory). SAMHD1 effectively suppresses caspase-1 activation and pyroptosis when infection occurs with cell-free virions. However, in the context of cell-to- cell transmission, which is 100-1,000-fold more efficient, SAMHD1 restriction is only partially effective resulting in the accumulation cytoplasmic viral DNA. This DNA is sensed by IFI16 resulting in caspase-1 activation and triggering of the pyroptotic death pathway. The action of other cellular factors like TREX1 and SLX4 (single and double strand DNA nucleases) may require further increase in the levels the cytosolic DNA needed to trigger pyroptosis. Conclusions: 1. The Vpx protein thwarts inflammatory pyroptosis following HIV-2 infection by degrading SAMHD1 thereby avoiding abortive infection and sensing of cytosolic viral DNA. 2. SAMHD1 is a bifunctional host factor capably restricting infection of resting CD4 T cells by cell-free HIV virions but functioning as a pro-pathogenic factor when resting CD4 T cells are infected by HIV-1 by the cell-to-cell route. 203 Mapping Vpx and Vpr Specificity in Antagonism of SAMHD1 Chelsea Spragg ; Michael Emerman Fred Hutchinson Cancer Research Center, Seattle, WA, US Background: The lentiviral accessory protein Vpx enhances infectivity of macrophages, dendritic cells, and resting T-cells by inducing degradation of the restriction factor SAMHD1, which blocks replication at reverse transcription. Vpx bridges SAMHD1 to the host ubiquitin ligase substrate receptor DCAF1, leading to polyubiquitination of SAMHD1 and degradation by the proteasome. The vpx gene is present in only two major lineages of lentivirus including HIV-2, but the paralagous vpr is present in all extant lineages. In certain cases, Vpr also has the ability to degrade host SAMHD1. SAMHD1 has evolved under positive selection due to viral antagonism, resulting in species-specificity between host SAMHD1 and viral Vpx/r. Depending on the lineage, Vpx exclusively targets either the N-terminus or the C-terminus of SAMHD1; however, the regions of Vpx/r controlling specificity are unknown. The structure of SIVmac Vpx bound to DCAF1 and the C-terminus of SAMHD1 has been solved, but there is extreme sequence diversity in vpr and vpx from divergent viruses. Methods: We used an evolutionary and structural approach to find appropriate and robust breakpoints in Vpx and Vpr in order to create functional, chimeric viral proteins. By assaying for the gain of ability to degrade resistant SAMHD1, these chimeric proteins assisted in mapping of determinants of specificity in Vpx and Vpr from several lentiviral lineages.

Poster Abstracts

203

CROI 2015