A Pan-HIV Strategy for Complete Genome Sequencing

Berg, Michael G.; Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert; Plantier, Jean‐Christophe; Brennan, Catherine A.

doi:10.1128/jcm.02479-15

Cited by 45 publications

(46 citation statements)

References 52 publications

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“…In contrast, O-Vpus generally failed to show significant activity against human tetherin. In this study, we confirmed that most HIV-1 O strains use Nef to counteract tetherin but also identified an exception and show that the Vpu protein of RBF206, an HIV-1 O strain isolated from a 47-year-old woman in France (37), is a potent antagonist of human tetherin. RBF206 Vpu and Nef both display anti-tetherin activity in transient-transfection assays and in the context of the HIV-1 M NL4-3 molecular clone.…”

supporting

confidence: 74%

“…An infectious molecular clone was constructed using unique BstEII (nucleotide position 2121), DraIII (nucleotide position 4152), and NheI (nucleotide position 8067) restriction enzyme sites and inserted between the MluI and NotI restriction sites of the pSMART-topo vector. Notably, the sequence of this RBF206 clone differs from a second independently derived consensus sequence of the same isolate, which was generated after an additional PBMC passage (37), by four nucleotides; however, none of these changes fell within the vpu gene, which is identical between the two consensus sequences. The pSMART-topo vector is a derivative of pSMART-LC-Amp containing the multiple-cloning site of pCR-XL TOPO from MluI to NotI.…”

Section: Methodsmentioning

confidence: 91%

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Efficient Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain

Mack

Starz

Sauter

et al. 2017

J Virol

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Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. In this study, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient-transfection assays, mainly Vpu promotes RBF206 release in infected CD4 T cells. Although mutations distinct from the adaptive changes observed in group M Vpus (M-Vpus) were critical for the acquisition of its anti-tetherin activity, RBF206 O-Vpu potently suppresses NF-κB activation and reduces CD4 cell surface expression. Interestingly, RBF206 Vpu counteracts tetherin in a largely species-independent manner, degrading both the long and short isoforms of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present the first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal. Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here we report an unusual exception, identifying a strain of HIV-1 group O (RBF206) whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoforms of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms.

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supporting

confidence: 74%

Section: Methodsmentioning

confidence: 91%

Efficient Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain

Mack

Starz

Sauter

et al. 2017

J Virol

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“…Complete genomes of HIV and co-infecting viruses can be obtained directly from patient specimens, provided viral loads are high enough (Luk et al, 2015 ; Yamaguchi et al, 2017 ). To increase sensitivity, the HIV-SMART method utilizes reverse primers in conserved sequences of HIV fused to a tag to facilitate combined virus-specific priming and amplification (Berg et al, 2016 ; Rodgers et al, 2017a ). In an alternate HIV-specific approach, individual long fragments (e.g., >2 kb) amplified with HIV primer pairs can be converted to NGS libraries to achieve detection limits nearing that of PCR, although the process is more labor intensive (Gall et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Universal Target Capture of HIV Sequences From NGS Libraries

et al. 2018

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Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens.Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software.Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads 90% for 35/40 samples with higher viral loads.Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

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“…of clinical variables to transmission clusters, it is ideal to characterize within patient viral variation as individual sequence variants (haplotypes) instead of combining all of the individual reads into a single consensus sequence 94,95 . Therefore, haplotypes for each patient were predicted from the sequence data using HAPHPIPE's haplotype stages.…”

Section: Haplotype Reconstruction For the Identification Of Transmismentioning

confidence: 99%

A cross-sectional study to characterize local HIV-1 dynamics in Washington, DC using next-generation sequencing

Gibson

Jair

Castel

et al. 2020

Sci Rep

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the DC Cohort Executive Committee † Washington, DC continues to experience a generalized HIV-1 epidemic. We characterized the local phylodynamics of HIV-1 in DC using next-generation sequencing (NGS) data. Viral samples from 68 participants from 2016 through 2017 were sequenced and paired with epidemiological data. Phylogenetic and network inferences, drug resistant mutations (DRMs), subtypes and HIV-1 diversity estimations were completed. Haplotypes were reconstructed to infer transmission clusters. Phylodynamic inferences based on the HIV-1 polymerase (pol) and envelope genes (env) were compared. Higher HIV-1 diversity (n.s.) was seen in men who have sex with men, heterosexual, and male participants in DC. 54.0% of the participants contained at least one DRM. The 40-49 year-olds showed the highest prevalence of DRMs (22.9%). Phylogenetic analysis of pol and env sequences grouped 31.9-33.8% of the participants into clusters. HIV-TRACE grouped 2.9-12.8% of participants when using consensus sequences and 9.0-64.2% when using haplotypes. NGS allowed us to characterize the local phylodynamics of HIV-1 in DC more broadly and accurately, given a better representation of its diversity and dynamics. Reconstructed haplotypes provided novel and deeper phylodynamic insights, which led to networks linking a higher number of participants. Our understanding of the HIV-1 epidemic was expanded with the powerful coupling of HIV-1 NGS data with epidemiological data.Despite recent reductions in HIV-1 prevalence in Washington, DC from 2.5% in 2013 1 to 1.8% in 2018, the United States (US) capital is still experiencing a generalized HIV-1 epidemic -as defined by the World Health Organization 2-4 . There were 340 newly diagnosed cases in DC in 2018, and the DC rate is five times higher than the national rate 3 . Blacks, men, men who have sex with men (MSM), and heterosexuals (HRH) account for the majority of people living with HIV-1 (PLWH) in DC 2,3 . However, ~20% of the newly diagnosed persons had an unknown risk for transmission in both 2016 and 2017 2,3 . Furthermore, the leading group (33.3%) of newly diagnosed cases was between the ages of 20-29 years old 3 . This same age group had the highest percentage (27.5%) of drug resistance mutations (DRM) at diagnosis, suggesting broader spread of HIV-1 drug resistant variants and potential concern for future therapeutic options, especially if these mutations are against first line antiretroviral (ART) drugs for newly infected individuals. With blacks and young adults being the most impacted groups of individuals for HIV-1 in DC, understanding the current HIV-1 phylodynamics can provide informative data to guide programs that prevent and reduce the incidence of HIV-1. Moreover, identifying potential transmission clusters amongst individuals in DC and their associated epidemiological features may help infer otherwise 'unknown' transmission modes and provide insight for more targeted prevention and intervention strategies.In 2011, the DC Cohort, a longitudinal observational NIH-funded co...

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A Pan-HIV Strategy for Complete Genome Sequencing

Cited by 45 publications

References 52 publications

Efficient Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain

Efficient Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain

Universal Target Capture of HIV Sequences From NGS Libraries

A cross-sectional study to characterize local HIV-1 dynamics in Washington, DC using next-generation sequencing

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