CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: A recently developed single virus imaging technique enables us to study IN (IN-eGFP) in individual viral complexes during early stages of the replication cycle. Different parameters including the number of fluorescent viral complexes, their distance to the nuclear envelope and intensity can be determined from these experiments. Although we can now identify single viral complexes in infected cells, we are unable to identify whether they contain reverse transcribed DNA. For this purpose, we combined our existing assay with the labelling of viral DNA using click chemistry. After addition of ethynyl- functionalised nucleosides during infection, these molecules are incorporated into the viral DNA by RT. The ethynyl-functional group on the nucleosides allows covalent linkage with azide reactive fluorophores via a copper-catalyzed azide-alkyne cycloaddition. It was shown earlier that HIV cDNA can be visualized by incorporation of EdU. However, this technique has some limitations. First, there is a high off-target labelling due to the incorporation of the nucleoside analogues by the host mitochondrial DNA polymerase. Secondly, viral DNA in the nucleus can not be dicriminated due to the incorporation of the functionalized nucleoside by the host DNA polymerase. Therefore, the use is limited to non- diving cells such as monocyte-derived macrophages (MDM). To overcome these limitations we develop RT specific ethynyl-functionalised nucleosides that are not incorporated by the cellular DNA polymerases. Results: With our novel nucleoside analogues we lowered the off-target labelling of the cellular DNA and reached up to 2% co-localization of the total number of IN-eGFP with the viral DNA staining in HeLaP4 cells and even 10% in MDM. Conclusion: This technology will allows us to simultaneous detect viral cDNA, IN-eGFP and capsid during the course of infection for a precise kinetic analysis of reverse transcription, trafficking, uncoating and nuclear import. 188 HIV RT STRAND TRANSFER AND NONTEMPLATED BASE ADDITION ARE MECHANISTICALLY INDEPENDENT Joanna Luczkowiak , Tania Matamoros, Luis Menéndez-Arias Centro de Biología Molecular Severo Ochoa, Madrid, Spain Background: Strand transfer (ST) events occur twice during HIV reverse transcription, the first during minus-strand DNA synthesis and the second, when the plus-strand DNA is polymerized. Both events rely on RNase H cleavage and template homology. Previous studies have shown that template switching is favored by short unpaired 3´-end tails in the DNA, suggesting that RT’s nontemplated nucleotide addition (NTA) activity could promote ST. We used in vitro ST and NTA assays and a panel of HIV-1 RTs to determine whether increased ST associates with high NTA activity. Methods: Tested enzymes were the wild-type (WT) HIV-1 BH10 RT and mutant L92P; and the WT HIV-1 ESP49 (group O) RT and ten mutants containing the connection subdomain substitutions K358R/A359G/S360A (3M) alone, or in combination with F61A, K65R/E478Q, L92P, L92P/D443N, L92P/E478Q, V148I, T355A/Q357M, D443N or E478Q. RTs were expressed and purified by ionic exchange followed by affinity chromatography. ST activity was assessed in vitro with three synthetic oligonucleotides, including a radiolabeled DNA primer, a RNA donor, and a partially homologous DNA acceptor. NTA activity was measured with 32P-labeled blunt-ended DNA/DNA template-primers (T/Ps). Results: RTs containing RNase H-inactivating mutations (all those having D443N or E478Q) were devoid of ST activity, while mutants containing F61A or L92P had very low ST activity (<7.5%). RTs with the highest ST efficiencies were 3M_T355A/Q357M (34.9%), 3M_V148I (29.3%), 3M (28.1%), WT HIV-1 ESP49 (13.9%) and WT HIV-1 BH10 (8.6%). All RTs except those having L92P showed NTA activity, and their best substrate was dATP, followed by dGTP, dCTP and dTTP. The 3M_V148I RT had high ST activity but low NTA activity (kobs = 0.013 min-1). In contrast, WT HIV-1 BH10 RT incorporated dATP efficiently on blunt- ended T/Ps (kobs = 0.227 min-1), but had relatively low ST activity. Overall, we found no correlation between the ST and NTA activities of tested RTs (as determined with a Pearson r test). Conclusion: Our results demonstrate that ST and NTA are mechanistically independent, meaning that for a specific RT, high NTA activity is not necessarily associated with increased template switching. These data are expected to be helpful to understand HIV variability at putative recombination sites, while providing further insight towards the characterization of ST as a distinct target for antiretroviral intervention.

189 UBP43 (USP18) ABROGATES INTERFERON AND SAMHD1-MEDIATED RESTRICTION OF HIV-1 Edmund Osei-Kuffour 1 , Kerstin Schott 2 , Philipp A. Lang 1 , Karl S. Lang 3 , Renate König 2 , Dieter Häussinger 1 , Carsten Münk 1 1 Heinrich Heine University Hospital, Düsseldorf, Germany, 2 Paul Ehrlich Institut, Langen, Germany, 3 University Hospital of Duisburg-Essen, Essen, Germany Background: Host intrinsic innate immune system directs antiviral defenses and viral restriction that includes, production of soluble factors such as type I and III interferon (IFN) and activation of restriction factors including SAMHD1. Ubiquitin-like protease 43 (USP18), negatively regulate IFN I and III by abrogating their signaling. SAMHD1 strongly inhibits HIV-1 replication in myeloid cells, however, it is efficiently antagonized by the HIV-2/SIV VPX. SAMHD1’s antiviral function in non-cycling cells is negatively regulated by phosphorylation at residue T592 by cyclin A2/CDKs. Intrigued by the recent model of USP18 enforced viral replication in murine macrophages (Honke et al. 2011), we asked whether human USP18 would be a factor influencing HIV-1 replication. Methods: THP-1 macrophage-like cells were generated that express USP18 or active site mutants. USP18 and SAMHD1 knockout THP-1 cells were generated by CRISPR-CAS system. Undifferentiated and PMA-differentiated THP-1 cells were infected by HIV-1 and -2 (+/- vpx ) luciferase reporter viruses. The infection was monitored by their luciferase activity and by qRT-PCR. Immunoblots analyzed the cellular expression of USP18, SAMHD1 and phospho-SAMHD1. The interaction of SAMHD1 with USP18, cyclin A, CKD1/2 and SKP2 was analyzed by pull-down assays and also visualized by confocal microscopy. The effect of USP18 on the cell cycle and dNTP pool was analyzed by flow cytometry and by biochemical assays respectively. HIV-1 mediated IFN induction was analyzed by qRT-PCR. Results: PMA-differentiated THP-1 cells are resistant to HIV-1 infection due to unphosphorylated SAMHD1 expression. This restriction is ablated when USP18 is expressed. USP18 expression in differentiated THP-1 cells enhanced HIV-1 infection by more than 40-fold and HIV-2Δ vpx by over 7-fold. HIV-1 infectivity rose by more than 100-fold in SAMHD1 knockout cells expressing USP18. Knockout of USP18 abrogated the infection of HIV-1 by more than 20 folds. SAMHD1 bound to USP18 in a complex with cylin A and CDK1/2. Also, USP18 interacted with SKP2, retained cylin A and down regulated p21, which induced phosphorylated SAMHD1 in differentiated THP-1.USP18 cells. USP18 abrogated IFN induction in the THP-1 cells, which correlated with higher HIV-1 infectivity. These activities of USP18 were independent of its ISG15 isopeptidase activity. Conclusion: This data provides evidence of direct involvement of USP18 in IFN and SAMHD1-mediated restriction of HIV-1. 190 VPR ENHANCES VIRAL GENE EXPRESSION FROM UNINTEGRATED DNA IN A DCAF1-DEPENDENT MANNER Sarah B. Nodder , Caitlin Miller, Hisashi Akiyama, Chelsea R. Ettinger, Rahm Gummuluru Boston University, Boston, MA, USA Background: While integration of HIV-1 DNA is essential for establishment of productive infection, unintegrated HIV DNA (uDNA) forms have been observed in several cell types, including resting CD4+ T cells, macrophages and dendritic cells (DCs), and are reported to be transcriptionally active. Expression of viral proteins from uDNA might contribute to virus-induced cytopathicity and immune activation, thus influencing virus reservoir in vivo. In this study, we demonstrate that virion-associated Vpr is required for optimal viral gene expression from uDNA. Methods: HeLa cells, PMA-treated THP-1 cells (THP1/PMA), primary macrophages, or DCs were infected with luciferase (in place of nef) expressing, VSV-G pseudotyped single cycle HIV-1 (incorporating wild type Vpr or W54R, Q65R, H71R and R90K-Vpr mutants) in the presence or absence of raltegravir (Ral) or with viruses containing a mutation in the catalytic domain of integrase (D116N). Alternatively, cells were infected with GFP-expressing single cycle HIV-2, SIVmac, and SIVsmm (±Vpr) in the presence or absence of Ral. Total viral DNA, extrachromosomal 2-LTR circles and integrated proviruses was measured by quantitative PCR. Extent of virus gene expression from uDNA was determined by quantifying luciferase expression in cell lysates, GFP expression by FACS, or p24gag expression by ELISA at variable times post infection. Results: Robust luciferase expression from uDNA (infections in the presence or Ral or infection with D116N integrase mutant) was only observed in the presence of Vpr. Furthermore, expression from uDNA in cells infected with Vpr-

Poster Abstracts

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CROI 2018