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Genetic variability of the U5 and downstream sequence of major HIV-1 subtypes and circulating recombinant forms.

ABSTRACT: The critical role of the regulatory elements at the 5' end of the HIV-1 genome in controlling the life cycle of HIV-1 indicates that this region significantly influences virus fitness and its biological properties. In this study, we performed a detailed characterization of strain-specific variability of sequences from the U5 to upstream of the gag gene start codon of diverse HIV-1 strains by using next-generation sequencing (NGS) techniques. Overall, we found that this region of the HIV-1 genome displayed a low degree of intra-strain variability. On the other hand, inter-strain variability was found to be as high as that reported for gag and env genes (13-17%). We observed strain-specific single point and clustered mutations in the U5, PBS, and gag leader sequences (GLS), generating potential strain-specific transcription factor binding sites (TFBS). Using an infrared gel shift assay, we demonstrated the presence of potential TFBS such as E-box in CRF22_01A, and Stat 6 in subtypes A and G, as well as in their related CRFs. The strain-specific variation found in the sequence corresponding at the RNA level to functional domains of the 5' UTR, could also potentially impact the secondary/tertiary structural rearrangement of this region. Thus, the variability observed in this 5' end of the genomic region of divergent HIV-1 strains strongly suggests that functions of this region might be affected in a strain-specific manner. Our findings provide new insights into DNA-protein interactions that regulate HIV-1 replication and the influence of strain characterization on the biology of HIV-1 infection.

SUBMITTER: Mbondji-Wonje C

PROVIDER: S-EPMC7411029 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Genetic variability of the U5 and downstream sequence of major HIV-1 subtypes and circulating recombinant forms.

Mbondji-Wonje Christelle C Dong Ming M Zhao Jiangqin J Wang Xue X Nanfack Aubin A Ragupathy Viswanath V Sanchez Ana M AM Denny Thomas N TN Hewlett Indira I

Scientific reports 20200806 1

The critical role of the regulatory elements at the 5' end of the HIV-1 genome in controlling the life cycle of HIV-1 indicates that this region significantly influences virus fitness and its biological properties. In this study, we performed a detailed characterization of strain-specific variability of sequences from the U5 to upstream of the gag gene start codon of diverse HIV-1 strains by using next-generation sequencing (NGS) techniques. Overall, we found that this region of the HIV-1 genome ...[more]

PMID: 32764600

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Project description:IntroductionThe latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype-tolerance using digital PCR.MethodsA subtype-B-specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype-tolerance in digital PCR (Bio-Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide.ResultsHIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty-nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG.ConclusionsThe results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non-B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

Project description:Intersubtype recombinants classified as circulating recombinant forms (CRFs) or unique recombinant forms (URFs) have been shown to play an important role in the complex and dynamic Brazilian HIV/AIDS epidemic. Previous pol region studies (2003-2013) in 828 patients from six states from Central Western, Northern and Northeastern Brazil reported variable rates of BF1, F1CB, BC, and CF1 mosaics. In this study HIV-1 subtype diversity BF1, F1CB, BC, and CF1 recombinants in pol were analyzed. Full/near-full/partial genome sequences were generated from F1CB and BF1 recombinants. Genomic DNA extracted from whole blood was used in nested-PCR to amplify four overlapping fragments encompassing the full HIV-1 genome. Phylogenetic trees were generated using the neighbor-joining/NJ method (MEGA 6.0). The time of the most recent common ancestor (TMRCA) of F1CB and BF1 clades was estimated using a Bayesian Markov Chain Monte Carlo approach (BEAST v1.8; BEAGLE). Bootscanning was used for recombination analyses (Simplot v3.5.1); separate NJ phylogenetic analysis of fragments confirmed subtypes. The phylogenetic analyses of protease/reverse-transcriptase sequences in 828 patients revealed 76% subtype B (n = 629), 6.4% subtype C (n = 53), 4.2% subtype F1 (n = 35), 13.4% intersubtype recombinants: 10.5% BF1 (n = 87), 2.3% BC (n = 19), 0.4% F1CB (n = 3), and 0.2% CF1 (n = 2). Two full and one partial BF1C genomes allowed the characterization of the CRF81_cpx that has 9 breakpoints dividing the genome into 10 subregions. Basic Local Alignment Search Tool searches (Los Alamos HIV Sequence Database) identified six other sequences with the same recombination profile in pol, five from Brazil, and one from Italy. The estimated median TMRCA of CRF81_cpx was 1999 (1992-2003). CRF60_BC-like sequences, originally described in Italy, were also found. Two full and one near full-length BF1 genomes led to the characterization of the new CRF99_BF1 that has six recombination breakpoints dividing the genome into seven subregions. Two new URFs BF1, with six recombination breakpoints and seven subregions were also characterized. The description of the first Brazilian BF1C CRF81_cpx and of the new CRF99_BF1 corroborate the important role of CRFs in the HIV/AIDS epidemic throughout Brazil. Our data also highlight the value of HIV-1 full-genome sequence studies in order to fully reveal the complexity of the epidemic in a huge country as Brazil.

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Genetic variability of the U5 and downstream sequence of major HIV-1 subtypes and circulating recombinant forms.

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Genetic variability of the U5 and downstream sequence of major HIV-1 subtypes and circulating recombinant forms.

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