CROI 2018 Abstract eBook

Abstract eBook

Oral Abstracts

Methods: We examined the inhibitory function of widely divergent SERINC5 proteins from different vertebrate species. To identify determinants responsible for the responsiveness of SERINC5 to Nef, we generated chimeras between Nef-sensitive and Nef-resistant SERINCs. We examined their ability to inhibit HIV-1 infectivity, their incorporation into HIV-1 virions, and their cell surface expression in the absence or presence of Nef. Results: We find that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. For instance, human and frog SERINC5 inhibited HIV-1 infectivity with similar potency, but frog SERINC5 was resistant to HIV-1 Nefs from different clades. Furthermore, our results indicate that sensitivity to Nef is, at least in part, governed by the fourth intracellular loop of SERINC5. A Nef-resistant SERINC5 became Nef-sensitive when intracellular loop 4 was replaced by that of human SERINC5. Conversely, human SERINC5 became resistant to Nef when its intracellular loop 4 was replaced by that of a Nef-resistant SERINC protein. We previously showed that HIV-1 SF2 Nef selectively inhibits the incorporation of SERINC5 but not of SERINC3 into progeny virions. Thus, we replaced the fourth internal loop of human SERINC3 by that of human SERINC5, and found that HIV-1 SF2 Nef strongly inhibited the incorporation of the resulting chimera into HIV-1 virions. In general, the fourth intracellular loop from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. Conclusion: Taken together, our results identify a major determinant of Nef responsiveness, and establish that human SERINC5 can be made to restrict HIV-1 infectivity even in the presence of Nef. 117 PROTECTIVE GENE EXPRESSION SIGNATURE IN RESPONSE TO RHCMV/SIV VACCINE VECTORS Connor Driscoll 1 , Richard Green 1 , Fredrik Barrenas 2 , Jean Chang 1 , Elise Smith 1 , Lynn Law 1 , Scott Hansen 3 , Louis J. Picker 3 , Michael Gale 1 1 University of Washington, Seattle, WA, USA, 2 Uppsala University, Uppsala, Sweden, 3 Oregon Health and Sciences University, Portland, OR, USA Background: The simian immunodeficiency virus (SIV)-targeted vaccine vectors based on rhesus cytomegalovirus (RhCMV) strain 68-1 harboring deletion of viral open-reading frames UL128 and UL130 elicit potent cellular immune responses that fully protect rhesus macaques (RM) against SIV infection through T cell-mediated viral clearance. When these viral ORFs were restored, the resulting 68-1.2 vaccine vectors failed to protect RM against SIV challenge. To define the molecular features of the protective 68-1 vaccine response, we interrogated and compared the transcriptomic host response from five groups of RMs: two groups vaccinated with 68-1 (subcutaneous or oral delivery), one group with 68-1.2 (subcutaneous delivery), one group with 68-1.2 DUL128 (subcutaneous delivery), and one group with a combination of 68-1 and 68-1.2 (subcutaneous delivery). Methods: Following vaccination, animals were subjected to repeated limiting dose intrarectal SIVmac239 challenge until infected by either detection of plasma virus or de novo development of T cell responses to SIVvif. Regardless of delivery mode, slightly over half of the animals that received the 68-1 vaccine manifested stringent aviremic control of the virus. There was no such control in any animal that received only a version of the 68-1.2 vaccine. Transcriptomic analysis (mRNA-seq) was performed on blood samples from all groups obtained during the vaccination phase. Bioinformatics analyses compared the transcriptional profiles between the protected and non-protected animal groups. Results: These analyses identified gene expression changes as early as three days after the first vaccination that distinguish protected from non-protected animals. Specifically, differences in the RNA profiles between protected and non-protected animals included magnitude and directionality of differentially expressed genes involved in several innate immune networks including innate immune activation, inflammation, and immune programming. Conclusion: These defined gene signatures for both protected and non- protected animals are being used to guide efforts to 1) understand the mechanisms responsible for the unique “control and clear” efficacy manifested by the 68-1 RhCMV vectors, 2) define intracellular response pathways of protection, 3) develop a modified vaccine that further enhances these features to achieve efficacy beyond the current ~55%, and 4) translate these vectors from nonhuman primates to people for protection against HIV infection.

1000 Genome Project ; 4) We quantified, the linkage disequilibrium in 120 kb A3 locus. Results: By analyzing A3 SNPs, RNAseq, and hypermutated viral sequences from thousands of HIV-1 patients and healthy donors, we have generated three independent datasets that indicate the source of skewed hypermutation patterns is natural genetic variations in A3G and A3H. First, only one hypermutation signature predominates in most clinical HIV-1 isolates. Second, A3G and A3H form two continuous haplotype blocks as a result of strong genetic linkage. Block 1 is prevalent outside Africa (particularly Asia) and contains the hypo-functional A3H HapI (GKE). Block 2 is prevalent in Africa and contains the hyper-functional A3H HapII (RDD). Third, A3H HapI and HapII and their respective A3G haplotypes a-g-t-t-t and g-c-c-c-c are expressed differentially. Conclusion: Overall, these results indicate that A3G and A3H are expressed differentially in different human populations and that these enzymes are the main sources HIV-1 hypermutation. The mutually exclusive function of A3G and A3H may be a source of weakness in our immunity to HIV-1. 115 HIV-1 SPLICING SUPPRESSION AS A POSSIBLE THERAPEUTIC TARGET Ann Emery 1 , Matthew Takata 2 , Jeffery Levengood 3 , John Collins 4 , Alice Telesnitsky 4 , Blanton Tolbert 3 , Paul Bieniasz 2 , Ronald Swanstrom 1 1 University of North Carolina Chapel Hill, Chapel Hill, NC, USA, 2 The Rockefeller University, New York, NY, USA, 3 Case Western Reserve University, Cleveland, OH, USA, 4 University of Michigan, Ann Arbor, MI, USA Background: HIV-1 splicing produces more than 50 alternatively spliced RNAs. The initial product is a full-length transcript and all other RNAs are made by splicing. Short 1.8 kb mRNAs remove all introns, but long 4 kb mRNAs retain the env intron. Unspliced mRNAs retain all introns. Transcripts with introns require Rev for nuclear export. Splicing has been studied as a possible theraputic target, but transmitted/founder viruses tolerate wide variation among spliced transcript levels. HIV-1 is unique in that the majority of transcripts retain introns, and viral infection was shown to cause intron retention in transcripts of cellular genes (Sherrill-Mix 2015). This suppression of splicing is unique to HIV-1 and thus a potential theraputic target. We present evidence of early and complete splicing suppression, mutations that override suppression, and possible roles of Rev in splicing suppression. Methods: Our Primer ID tagged deep sequencing assay quantifies HIV-1 spliced transcripts within the two RNA size classes. A second assay uses a Primer ID- tagged random reverse primer coupled with a fixed forward primer upstream of the major splice donor to detect changes in the amounts of full-length, long spliced transcripts (containing the env intron), and short fully spliced transcripts. Results: Mutations to exonic splicing silencers activated the upstream proximal acceptor site. They also lifted suppression of splicing (i.e. activated splicing) at the major 5’ splice donor D1 but not at splice donor D4. Activating mutations were made both to known silencers, and novel silencer elements were discovered by synonymous mutagenesis of the HIV-1 genome. We observed that the absence of Rev decreases but does not eliminate unspliced or partially spliced transcripts. We looked for but did not find evidence of transcripts that fail to use D1 (retain the gag/pro/pol intron) but use D4 to splice out the env intron. Additionally, we found no transcripts that used D2 or D3 without first using D1, making D1 suppression a marker for suppression of all splice donors. Conclusion: Silencer elements that activate acceptor sites can also lift suppression of splicing from D1. As splicing is cotranscriptional and efficient, the absence of spliced gag/pro/pol transcripts suggests unspliced transcripts are suppressed early and completely from all splicing, long before the other downstream splice donors or the Rev Response Element (RRE) are transcribed. The role of Rev in splicing suppression remains to be fully studied. 116 A LONG INTERNAL LOOP GOVERNS THE SENSITIVITY OF THE ANTI-HIV PROTEIN SERINC5 TO NEF Weiwei Dai , Yoshiko Usami, Yuanfei Wu, Heinrich Gottlinger University of Massachusetts, Worcester, MA, USA Background: We have recently identified the multipass transmembrane proteins SERINC3 and SERINC5 as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. However, the determinants that govern the sensitivity of SERINCs to Nef remain unknown.

Oral Abstracts

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CROI 2018