Rapid Sequencing of Complete<i>env</i>Genes from Primary HIV-1 Samples

Smith, Melissa; Murrell, Ben; Eren, Kemal; Ignacio, Caroline; Landais, Elise; Weaver, Steven; Phung, Pham; Ludka, Colleen; Hepler, Lance; Caballero, Gemma; Pollner, Tristan; Guo, Yan; Richman, Douglas D.; Poignard, Pascal; Paxinos, Ellen E.; Pond, Sergei L. Kosakovsky; Smith, Davey M

doi:10.1093/ve/vew018

Cited by 31 publications

(42 citation statements)

References 25 publications

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“…Although a recent study that sequenced the envelope genes of two different HIV clones estimated that the PCR recombination rate was only 0.46 to 0.85% (37), in vitro RT and PCR recombination rates of ϳ 30% were reported previously (22,38). In our experimental setup, we found a similarly high recombination rate.…”

Section: Discussionsupporting

confidence: 59%

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Raghwani

Koekkoek

et al. 2017

J Virol

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In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottle-

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Section: Discussionsupporting

confidence: 59%

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Raghwani

Koekkoek

et al. 2017

J Virol

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“…5 and Supplementary Table 8) 51 . Frequencies of detected variants carrying a 325 or 332–334 mutation agreed well among SMRT sequencing, PIDS, and SGS (Supplementary Table 8).…”

Section: Resultsmentioning

confidence: 99%

“…Full-length HIV-1 env was amplified and sequenced on the Pacific Biosciences RS-II as described previously with two minor modifications 51 . First, primers were designed specifically for sequenced subjects using available SGS sequences that spanned the primer regions.…”

Section: Methodsmentioning

confidence: 99%

“…First, primers were designed specifically for sequenced subjects using available SGS sequences that spanned the primer regions. Primers used for amplification were 5′-GAGCAGARGACAGTGGCAATGA-3′ and 5′-GGAGAATAGCTCTACGAGCTCTTTG-3′ for 1HB3, 5′GAGCAGAAGACAGTGGCAATGA-3′ 51 and 5′TGACCATTTATTGCCCATCTTATAGC-3′ for 1HC2 and 5′-GAGCAGAAGACAGTGGCAATGA-3′ 51 and 5′-CCACTTGCCACCCATTTTATAGGA-3′ for 1HD1. Second, the more recent PacBio P6-C4 chemistry was used.…”

Section: Methodsmentioning

confidence: 99%

“…Data was pre-processed using the PacBio CCS2 algorithm, generating single molecule circular consensus sequences. The Full-Length Envelope Analysis (FLEA) pipeline 51 was used for further processing and visualization. MAFFT 65 with manual curation was used to align amino acid sequences, and fastree2 66 was used to infer maximum likelihood phylogenies.…”

Section: Methodsmentioning

confidence: 99%

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Antibody 10-1074 suppresses viremia in HIV-1-infected individuals

Caskey¹,

Schoofs²,

Gruell³

et al. 2017

Nat Med

Self Cite

425

507

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Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-1 envelope protein. It is among the most potent anti-HIV-1 neutralizing antibodies isolated to date. Here we report on its safety and activity in 33 subjects who received a single intravenous infusion of the antibody. 10-1074 was well tolerated with a half-life of 24.0 days in uninfected and 12.8 days in HIV-1-infected subjects. 13 viremic subjects received the highest dose of 30 mg/kg 10-1074. 11 of these participants were 10-1074-sensitive and showed a rapid decline of viremia by a mean of 1.52 log10 copies/ml. Virologic analysis revealed the emergence of multiple independent 10-1074-resistant viruses within the first weeks after infusion. Emerging escape variants were generally resistant to the related V3-specific antibody PGT121, but remained sensitive to antibodies targeting non-overlapping epitopes, such as the anti-CD4 binding site antibodies 3BNC117 and VRC01. The results demonstrate the safety and activity of 10-1074 in humans and support the idea that antibodies targeting the V3 glycan supersite may be useful for treatment and prevention of HIV-1 infection.

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Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues

et al. 2018

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Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

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Rapid Sequencing of CompleteenvGenes from Primary HIV-1 Samples

Cited by 31 publications

References 25 publications

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Antibody 10-1074 suppresses viremia in HIV-1-infected individuals

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues

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