The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

Ethical approval for the present study was granted by the Institutional Review Board of YRG CARE (Y. R. Gaitonde Centre for AIDS Research and Education), Chennai. A written informed consent was obtained from all the subjects enrolled in the study and maintained with confidence. The Human Ethics and Biosafety Committee of JNCASR (Jawaharlal Nehru Centre for Advanced Scientific Research), Bangalore reviewed the proposal and approved the study.

From the clinical records available at YRG CARE over the past several years, seropositive subjects were enrolled in two different clinical cohorts, called the YRG CARE (Chennai, Tamil Nadu) and Nellore (Andhra Pradesh) cohorts, for the longitudinal study spanning over a period of 2–3 years. 30 subjects belonged to YRG CARE cohort, Chennai, Tamil Nadu and 35 subjects to the Nellore cohort, Andhra Pradesh. The participants of the YRG CARE cohort were diagnosed between years 1996 and 2008, and the dates of diagnosis of the Nellore cohort are not available. The clinical samples were collected at an interval of 6 months. The primary inclusion criteria consisted that the CD4 counts should be above 500 cells/μl at the time of enrollment and that the subjects should be drug-naive and free of AIDS-related clinical symptoms. The exclusion criteria included the presence of opportunistic infections, signs of acute systemic illness and prior antiretroviral treatment. The study participants consisted of only adult subjects over 18 years of age. The subjects were all drug-naive and believed to have acquired the infection primarily through heterosexual transmission. The clinical profile of the subjects including the subject ID, sampling date, age, gender, viral load and CD4 count has been summarized (Table 1).

A single vial of 20 ml of peripheral blood was collected from each participant at 6-month intervals during 2010–13. The blood samples were collected in EDTA vacutainers (Becton Dickinson, San Diego, USA) and were processed on the same day of collection. The PBMC and plasma samples were stored in 1 ml aliquots in a liquid nitrogen container and a deep freezer, respectively. Genomic DNA extracted from 200 μl of whole blood using a commercial DNA extraction kit (QIAmp Blood Mini Kit, Cat. No: 69504, Qiagen India, New Delhi, India). The CD4 T-cell count was determined using the BD FACSCount Reagent Kit (Cat. No: 340167, Becton Dickinson, San Jose, California, USA) and the BD FACSCount Control Kit (Cat. No: 340166) following the manufacturer’s instructions. The samples were analyzed on a BD FACS Calibur flow cytometer. The plasma viral RNA load was determined using the Abbott m2000rt Real-Time PCR machine (Abbott Molecular Inc. Des Plaines, IL, USA). All the methods were carried out in accordance with Institutional Ethics Committee for Human Research guidelines of the Indian Council for Medical Research (ICMR), New Delhi.

RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany). In the case of the clinical samples that failed the PCR, an alternative kit was used to extract the viral RNA from 1 ml of plasma (the NucliSENS miniMAG nucleic acid extraction kit, Ref. No. 200293, BioMerieux, France). The complementary DNA (cDNA) was synthesized using random hexamers and a commercial kit (SuperScript® III Reverse Transcriptase, Cat. No: 18080–051, Invitrogen, Carlsbad, California, USA). The reaction vials were incubated at 25 °C for 10 min and 50 °C for 50 min. The reactions were terminated at 85 °C for 5 min followed by RNaseH treatment. The cDNA was used for the amplification of Gag and other viral gene segments.

The full-length gag was amplified from the cDNA using a nested-PCR strategy and a commercial long-range PCR kit (XT-20 PCR system, Merck Genie, India) on the I-Cycler (Bio-Rad, California, USA). Furthermore, from a select subset of the participants, Gag p6, LTR, Protease, RT, RNaseH, Integrase and Env regions were also amplified. The details of the amplification and sequencing primers are summarized for gag in Table 2 and the rest of the gene segments in Table 3. The sequence of the V3-V5 envelope region (0.7 kb fragment, 7001–7667, HXB2 coordinates) of a few select subjects was determined using the HIV-1 Env Subtyping Kit (The NIH AIDS Research & Reference Reagent Program, Germantown, MD, USA).

The reaction conditions for the Gag amplification in both the rounds of the nested PCR were 94 °C for 2 min for one cycle and then 35 cycles, each cycle consisting of melting at 94 °C for 30 s, annealing at 50 °C for 50 s, and extension at 72 °C for 2 min, followed by final extension at 72 °C for 5 min. Two μl of the first-round PCR products were transferred to the second-round PCR to amplify the complete gag sequence. The details of the primers and the amplified products are summarized in Table 2. Carryover contamination was prevented by adherence to strict procedural and physical safeguards that included reagent preparation and PCR setup, amplification, and post-PCR processing of samples in separate rooms. The PCR products were purified using a commercial DNA purification kit (Cat. No. YDF100, Real Biotech Corporation, Taiwan) and subjected to sequencing. The sequencing was performed on ABI PRISM® 3130xl Genetic Analyser (Applied Biosystems, Illinois, USA) using multiple internal sequencing primers. The sequences are available from GenBank under the accession numbers KF578465-KF578467, KT124420-KT124478, KT152633-KT152672, and KP890700-KP890762.

Phylogenetic analysis was performed using the reference sequences of HIV-1 group M available from the Los Alamos database (http://​www.​hiv.​lanl.​gov/​). Sequences were manually edited using Bio-Edit software version 7.0.5.3. Multiple sequence alignments were performed using ClustalW in the BioEdit software (version 7.0.5.3). The phylogenetic tree was constructed using the neighbor-joining method (Kimura two-parameter model) in 1,000 bootstrapped data sets, using the Molecular Evolutionary Genetics Analysis (MEGA) software version 5 [30]. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All the positions containing gaps and missing data were eliminated. The final dataset contained a total of 1,632 nucleic acid positions. Additionally, genetic subtype characterization of the gag sequences was performed using the REGA HIV-1 Subtyping Tool version 2.0, available at the BioAfrica site (http://​www.​bioafrica.​net/​rega-genotype/​html/​) and the recombinant identification tool available at the Los Alamos HIV Sequence Database (http://​hiv.​lanl.​gov/​content/​index). Every individual gene sequence was subjected to a BLAST analysis against the laboratory sequence database to confirm authenticity. All the other HIV-1 gag sequences required for the analysis were retrieved from the Los Alamos HIV Sequence Database (http://​hiv.​lanl.​gov/​content/​index).

To evaluate the global prevalence of the PTAP duplication, the Los Alamos National Laboratory (LANL) HIV sequence database was searched for global HIV-1 gag sequences for subtype A, B, C, D, F, G, CRF01_AE, CRF02_AG and CRF07/08_BC as of October 2015. Of note, the partial sequence insertions not duplicating the core PTAP motif or amino acid insertions not related to PTAP were not included in the analysis. In silico analysis was performed to understand the prevalence of PTAP duplication. Further, to evaluate the profile of PTAP duplication over the past 30 years, subtype B and C sequences were downloaded from the LANL sequence database excluding the problematic sequences and acquiring one sequence per patient. Of note, for many sequences, the information regarding the date of isolation of the clinical sample was not available from the database. We, therefore, considered the date of sequence submission instead for the analysis. It should be noted that there could be a significant delay between the actual date of sample collection and sequence submission. Amino acid sequences downloaded in the FASTA format were categorized into five groups representing five time periods, beginning with the year of the first sequence deposited till 2015. The regression analysis was performed using GraphPad Prism version 5.02 to examine the trend of PTAP motif duplication over the period of past 30 years.

In the present manuscript, we refer to all the viral strains that contain a complete PTAP motif duplication in Gag p6 as the double-PTAP strains and those that lack such duplication as the single-PTAP strains. The double-PTAP strains must contain a second and intact PTAP motif comprising of the four core amino acids with or without the additional flanking amino acids. To examine the nature of the PTAP sequence insertion in HIV-1 Gag p6 in different HIV-1 genetic subtypes, we analyzed a total of 17,769 full-length HIV-1 Gag sequences available from the Database. Eleven percent of the sequences (1,997) contained insertions that constituted the duplication of the PTAP motif. The analysis identified two important characteristics of subtype C. The frequency of the sequence duplication and the length of sequence duplication, both was found significantly higher in subtype C as compared to other subtypes (Fig. 2). Twenty-nine percent of the sequences (1,474 of 5,074) of subtype C contained the complete duplication of the PTAP motif (Fig. 2a). A considerably smaller proportion (380 of 5,901; 6.4%) of subtype B strains also contained a complete PTAP duplication. In contrast, only a low frequency of subtype D sequences (12 of 8,767; 0.1%) contained the PTAP duplication. All the other HIV subtypes collectively contained less than 5% of sequences characterized by the PTAP duplication. The BC recombinant HIV-1 strains represented 8% of sequences containing the PTAP duplication. The HIV-1 groups N, O, and P contained few sequences to permit a meaningful sequence analysis.

Furthermore, the length of the PTAP motif duplication was quite heterogeneous with some of the insertions being as long as 16 amino acid residues. To understand if the length of the sequence insertion has any association with the nature of viral subtype, the sequences of subtypes B and C were compared, as only these two subtypes contained sufficient numbers. In subtype C, while 7% (357/5,074) of Gag p6 sequences contained PTAP insertions 12–16 amino acids or longer, only 0.39% (20/5074) of the sequences contained sequence duplication of three amino acids or less (Fig. 2a). In contrast, in subtype B, only 1% of the sequences (52/5,901) contained PTAP insertions of 12–16 amino acids and as many sequences as 11.2% (665/5901) contained insertions of three amino acid or less. Collectively, the above analysis is indicative that subtype C has the propensity to introduce PTAP sequence duplications at a higher frequency, and the duplication length is typically longer.

Additionally, to understand if the prevalence of the PTAP duplication has been undergoing a variation over the years, we categorized the full-length gag sequences of subtypes B and C into 5-year phases starting from 1979 (subtype B) or 1986 (subtype C) up to 2015 (Fig. 2b). In subtype B, the percent prevalence of the PTAP duplication strains increased between phases 1996–2000 to 2001–2005 from 3.4 to 6.5% and remained stable after that. A comparable trend was seen in the context of subtype C but at a higher prevalence. Between phases 1996–2000 to 2001–2005, the percent prevalence of the PTAP duplication strains of subtype C increased from 17.6 to 25% and increased further to 31% prevalence in the final phase of 2010–2015 although the sample size for the last phase is indeed small. Using the regression analysis to understand the trend, we observed that the increasing trend of PTAP duplication in subtype C is statistically significant (p = 0.03) unlike in subtype B (p = 0.17) where no such trend was seen. Thus, unlike in subtype B where the prevalence of the PTAP duplication appears to have been stabilized at the global level, the prevalence of PTAP duplication appears to be increasing further in subtype C, which must be confirmed in future studies.

The sequence analysis performed above confirmed the global predominance of the PTAP motif duplication in subtype C Gag sequences. However, the disease status of the subjects, and their anti-retroviral therapy status and its influence on the PTAP motif duplication are not known. Additionally, the profile of the PTAP sequence duplication and its evolutionary stability has not been examined in a longitudinal study especially in the context of subtype C infection. To fill this gap, we monitored two different southern Indian clinical cohorts, consisting of 65 drug-naïve subjects; in a 2 to 3 years follow-up spanning 2010–2013 with a repeat blood sampling every 6 months. All the subjects belonged to the chronic stage of the viral infection and were infected with subtype C reportedly by heterosexual transmission. The clinical profile of all the 65 study subjects has been documented (Table 1).

Using a nested PCR, we could successfully determine the full-length gag sequence from 50 of the 65 subjects either from the plasma viral RNA or the genomic DNA extracted from whole blood. In a phylogenetic analysis, all the primary gag sequences clustered together and with the reference subtype C viral sequences confirming their phylogenetic identity (Fig. 3). Of the total 126 gag sequences generated from the 50 subjects, 100 sequences derived from 42 subjects lacked a sequence duplication of any kind, thus, representing the wild type single-PTAP gag profile (Fig. 4). The PTAP elements consisting approximately of 14 amino acid residues of the 100 Gag sequences were aligned (Fig. 4). Several of the sequences contained nucleotide insertions or deletions of variable length including single amino acid variations in different regions of Gag. In two subjects (T019 and 2010), sequence insertions were found at the N-terminal of Gag p6, but these duplications were not related to the PTAP motif. The PTAP motif duplication was found in the remaining eight of the 50 subjects (T004, T014, 2006, 2012, 2018, 2020, 2032, and 2037). Thirty-one sequences from the eight subjects were aligned in the PTAP region consisting of a 14 amino acid window (Fig. 5). A sequence of 14 amino acids (NRPEPTAPPAESFR, the four core residues underlined) representing subtype C consensus PTAP motif consisting of the four core residues and the ten flanking residues was used as references in the multi-sequence alignment. Several important observations could be made from the sequence analysis regarding PTAP duplication in the eight subjects. First, the length of sequence duplication was variable in the primary viral isolates. The length of the original PTAP motif (right side of the dashed line, Fig. 5) and that of the duplicated PTAP motif (left side of the dashed line, Fig. 5) both vary in the length of the sequences. For a simple representation of these differences in the PTAP motifs, we use a formula consisting of two figures. to represent each viral isolate. The right and left-hand numbers in the formula represent the number of amino acid residues in the original and duplicated PTAP motifs in Gag p6 of the viral isolate, respectively. Using this formula, we found that three of the eight subjects (T004, 2012, and 2032) contained an original PTAP motif of 14 residues and a duplication of 14 amino acids thus representing a configuration of 14 + 14. In contrast, subjects 2018 contained a PTAP configuration of 12 + 14 representing an original PTAP motif of complete 14 amino acid length but a duplicated PTAP motif shorter by two amino acids and 2020 contained a PTAP configuration of 11 + 14 representing a duplicated PTAP motif shorter by three amino acids. Subject 2037 contained a 9 + 11 configuration while the other two subjects T014 and 2006 an 8 + 12 configuration of PTAP duplication. Second, regardless of the differences constituting the original or duplicated PTAP motifs, in all the subjects at all the time points, the ‘core PTAP motif’ is invariably conserved. Additionally, the aspartic amino acid residue (E) located immediately upstream of the core motif is also invariably conserved in the original as well as the duplicated sequences alluding to the functional importance of these five (EPTAP) residues. Additionally, in an analysis of sequences downloaded from the extant database also, we observed that the upstream aspartic amino acid residue is highly conserved. Only 34 of 1,804 (1.8%) subtype C sequences demonstrated variation at this residue (single sequence per subject was included in the analysis, data not shown). The variations observed between the original, and the duplicated sequences were exclusively mapped to the other nine flanking residues of the 14 amino acid motif. The highly preserved ‘EPTAP’ motif is also seen in the viral strains containing a PTAP motif duplication of shorter length. Third, in all the viral strains, the duplicated PTAP motif typically demonstrates variation at least in one residue as compared to the original PTAP motif. Thus, a sequence variation between the original and duplicated PTAP motifs appears more common and necessary. Between the original and the duplicated PTAP motifs, the former is usually genetically more homologous to subtype C consensus sequence. Thus, the duplicated PTAP motif demonstrates a sequence variation of a higher order. Fourth and importantly, the length of the duplicated PTAP sequence remained constant at the follow-up time points in each subject suggesting the stability of the sequence length once the motif is duplicated. In subject T004, for instance, the sequence of the duplicated motifs remained constant consisting of 14 residues at all the six different time points spaced 6-month apart (M0, M6, M12, M18, M24, and M30). Additionally, the duplicated sequence may undergo further variation in the amino acid sequence (compare time points M0, M6 and M12 of subject T004. Likewise, different time points of subjects 2032 and 2020), but not in the length of the motif. This observation suggested that in the chronic phase of the viral infection, once the founder viral strains were established possibly in the acute phase, the length of the duplications remain stable although the sequence diversity within and between the duplicated and the original 14 amino acids is highly variable. Lastly, at least in two subjects (2018 and 2014), the presence of the single-PTAP as wells as double-PTAP viral strains was detected suggesting a possible mixed infection. Of note, the conventional PCR amplicon sequencing strategy employed here is limited by the inability to detect minority viral strains if they are not represented at least at a prevalence of 20% [31]. Thus, it remains a possibility that other subjects also might contain the single-PTAP viral strains, but these minority species were not visible in the conventional sequencing that we employed here. In summary, the PCR fragment sequence strategy identified that the original PTAP sequence was relatively conserved among the subjects, but when any part of the sequence comprising of the PTAP core motif is duplicated, the amino acid residues flanking the PTAP core demonstrated a higher magnitude of genetic variation.

To find a possible association between PTAP duplication and drug resistance, primary or transmitted, we determined the sequences of the pol sequence (Protease, RT, RNase H, and Integrase; coordinates 2,253 to 5,096 as in HXB2) of the viral strains from all the six subjects of our cohort containing PTAP duplications. We could successfully amplify the protease from all the six subjects (Accession numbers: KT124484-KT124490). The RT (Accession numbers: KT124479-KT124483) and Integrase (Accession numbers: KT428977-KT428981) regions could be amplified from only five subjects. The sequences were subjected to drug resistance analysis using the HIV drug resistance database at the Stanford University (http://​hivdb.​stanford.​edu/​). This analysis failed to find any mutations associated with drug resistance in any of the viral enzymes in these six subjects. Thus, this analysis, on the one hand, confirmed the drug-naïve status of the six subjects and on the other hand, ruled out ART as a cause for the appearance of PTAP duplication in these subjects. The immune pressure in these subjects could be a selection pressure that may have mediated the PTAP sequence duplication. To understand if PTAP duplication is associated with the sequence insertions in other regions of the viral strains, we determined the sequences of the LTR and env from the six subjects containing the PTAP duplication. Env and LTR could be amplified from four (ascension no: KP683336-KP683342) and five (ascension no: KP683343- KP683348) of the six subjects, respectively. We did not find any sequence insertions or other modifications in the V3 loop of the envelope or the viral promoter (data not shown).

As in India, the viral epidemic in South Africa is dominated by HIV-1 subtype C. We had access to the full-length gag sequences of the CAPRISA clinical cohort in the city of Durban and rural region of Vulindlela (the Acute Infection Study Team, CAPRISA, Durban), South Africa. The clinical profile and the study objectives of the cohort have been reported previously [32, 33]. The gag sequences of subtype C origin were derived from 75 subjects, constituting a subset of the CAPRISA 002 cohort. All the 75 subjects were drug-naïve, in the chronic phase of the infection, and recruited during 2004 to 2010. Access to the CAPRISA clinical cohort offered an opportunity to examine the prevalence and evolution of PTAP duplication in a different clinical cohort dominated by subtype C. A total of 150 subtype C p6 gag sequences, two from each subject, were available for the analysis. The first sequence from each subject was drawn from the acute phase of the infection at the time of screening or enrollment while the second sequence at a later time point eight to 25 months post-recruitment, representing the chronic phase of the infection. All the 150 p6 gag sequences were evaluated for sequence insertion of any type in the PTAP motif region of p6 and aligned with a subtype C consensus sequence generated using the sequences derived from the Los Alamos HIV Sequence Database, 2004 (Fig. 6). The analysis identified sequences of four different kinds in the 75 subjects depending on the nature of PTAP duplication. Of the 150 sequences, 64% (96/150) did not contain a sequence insertion of any kind in the PTAP motif region thus, representing the wild type profile. Of the rest of the 54 sequences, four sequences derived from two subjects (CAP008 and CAP224) lacked the duplication of the PTAP motif but contained an insertion of a sequence string located immediately upstream of the PTAP motif. Of the remainder of the 50 sequences, 42 sequences (28%, 42/150) contained a complete duplication of the PTAP core motif (Fig. 7). In the remaining eight sequences derived from four subjects (CAP188, CAP248, CAP308, and CAP357), the duplicated PTAP motif was mutated to PTAL, ATAP or PTTP motifs as also observed in the LANL database. Each of the mutated motifs differed from the PTAP motif at a single amino acid residue. It is not known if such variations are biologically functional. We did not find such variations in the duplicated PTAP motif in the Indian clinical cohort of the present study probably given the small sample size, only eight viral strains. The insertion length in the 42 sequences containing the complete PTAP duplication ranged from 4 to 16 amino-acids in the South African cohort. Importantly, of the 42 gag p6 sequences derived from 22 subjects that contained a complete PTAP duplication, in 20 subjects, the duplication was observed at both the time points of sample collection, at baseline and the follow-up time point. In two subjects (CAP211 and CAP229), the duplication was found only at the follow-up time point but not at baseline. Since a conventional Sanger sequencing strategy was used to examine the viral species in all these samples, it is not known if the non-detection of the PTAP variant viral strains at earlier time points from CAP211 and CAP229 could be ascribed to the limitations in sensitivity. Collectively, the data demonstrated a prevalence of 29.3% (22 of 75 subjects) PTAP sequence duplication in the drug-naïve population of the South African subtype C viral strains, thus, independently confirming our observations. The present analysis combined with that of the published reports of subtype C collectively ascertains that (1) there is significantly higher representation of the PTAP motif duplication in subtype C as compared to the other subtypes, (2) the mean length of the PTAP sequence duplication in subtype C is significantly larger.