The HIV-1 matrix protein p17 (p17MA) encompasses the amino (NH2)-terminal domain of the Gag gene,1 and in mature virions, it is a 131-amino acid polypeptide myristoylated at the NH2-terminus that forms a protective shell associated directly with the inner leaflet of the viral membrane.2 This pleiotropic viral protein serves several functions in HIV-1 replication cycle, is involved in nuclear import of proviral DNA,3,4 and targets Pr55Gag proteins to their assembly sites at the plasma membrane (PM).5,6 Mutational studies have shown that a large deletion in p17MA redirects HIV-1 assembly to the endoplasmic reticulum,7 whereas point mutations, particularly in the NH2-terminal highly polybasic region, shift the site of assembly from the PM to internal compartments8 defined as late endosomes or multivesicular bodies.9 The interaction of p17MA with PM is mediated by the myristoyl moiety and by a cluster of positively charged amino acid residues located in the NH2-terminal region of the protein8 that furthers its ability to associate with negatively charged phospholipid head groups.10,11 Interaction of p17MA with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] serves an important function in directing Pr55Gag to PMs,10 and p17MA binding to PI(4,5)P2 promotes both exposure of the p17MA myristoyl group and protein oligomerization,12 thus favoring virus assembly. In the late stage of infection, a key function of p17MA is the recruitment of the viral surface/transmembrane gp120/gp41 envelope protein complex into virions.1,13
The critical role of p17MA in regulating the virus life cycle is related to its capability of interacting with as many as 20 different cellular proteins.14,15 On the basis of the wealth of evidence obtained by several groups, p17MA has been long believed to have a highly conserved primary amino acid sequence and a well-preserved structural integrity to avoid severe fitness consequences. It is therefore likely that distinct sites, particularly those at the surface of the protein, are constrained due to these unavoidable vital interactions. It is worth noting that virions carrying NH2-terminal highly polybasic region mutations not only showed an early entry defect and a reduced capacity to integrate their proviral genomes16 but also displayed a severe impairment in their ability to incorporate envelope glycoproteins.17,18
However, recent data point to the carboxy (COOH)-terminus of p17MA as a highly flexible domain and with a high level of predicted intrinsic disorder,19 which would subtend to at least partially unfolded status of this region.20 Unlike the NH2-terminus, mutations in the p17MA COOH-terminal region were not found to affect viral replication and infectivity.6 Therefore, the p17MA COOH-terminus has to be considered evolutionarily relevant, as mutations affecting this region may generate unfolded proteins without resulting in severe fitness consequences. More importantly, as recently pointed by Giagulli et al,21 sequence variations in this region of p17MA could result in protein variants endowed with different functional properties. Elucidation of this issue is of pathogenic relevance considering that p17MA is released by HIV-1–infected cells,22 is detected in different organs and tissues even in patients under highly active antiretroviral therapy (HAART)23–25 and that, extracellularly, p17MA exerts pleiotropic effects on a variety of immune cells.24,26–30
With the aim to assess the degree of variations in the p17MA primary sequence, and particularly to identify domains or residues possibly affected by recurrent changes of potential functional relevance, ultra-deep pyrosequencing (UDPS) of p17MA was performed on plasma virions from 18 viremic HIV-1–infected patients. Here, we show a relatively high number of components of p17MA quasispecies in all patients investigated, with a certain degree of variability in intrapatient frequency. Notably, although mutations were scattered along the entire sequence of the viral protein, clusters of mutations were more frequently detected at the COOH-terminal region of p17MA. On the whole, our data show that the intrapatient level of sequence diversity in the p17MA is much higher than predicted before. The presence of p17MA quasispecies diversity may offer new tools to improve our understanding of the viral adaptation during the natural history of the infection.
MATERIALS AND METHODS
Eighteen consecutive residual plasma samples from HIV-1–infected patients undergoing routine monitoring of viremia were selected, with HIV-RNA values of at least 10,000 copies per milliliter. Clinical and virological characteristics of patients are shown in Table 1. Four patients (patients 3, 4, 16, and 18) were naive to antiretroviral therapy, 14 were experienced, but all were off therapy at the time of sampling, for various reasons. None of the patients presented an AIDS-defining condition. All patients were infected with HIV-1 subtype B, as determined by the sequence analysis of p17MA region, according to the Los Alamos genotyping algorithm. Before starting the analyses, the samples were completely anonymized, and the only remaining linkage was with the clinical data included in Table 1.
UDPS and Data Analysis
Plasma HIV-1 RNA was extracted and quantified by real-time polymerase chain reaction (PCR) (Abbott real time HIV; Abbott Molecular, Inc, Des Plaines, IL). p17MA amplification was performed by nested PCR. Briefly, 2 rounds of 30 cycles (94°C for 2 minutes, 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 68°C for 30 seconds, and final elongation at 68°C for 5 minutes) were carried out using a proofreading DNA polymerase (Platinum Taq DNA Polymerase High Fidelity; Invitrogen, by Life Technologies, Monza, Italy). The first round included a 1-step reverse transcriptase–polymerase chain reaction, using a Platinum quality proof reading reverse transcriptase (Invitrogen). Unique in house-designed stretches of 8 nucleotides (Multiplex Identifiers) were used to tag each sample. For each sample, the amplicons from at least 4 replicate PCRs were pooled, representing the content of 1 mL of plasma. According to this protocol, a minimum of 11,200 templates were analyzed by UDPS, on the basis of viral load (Table 1), minimizing the occurrence of artefacts attributable to template resampling.31 UDPS was carried out with the 454 Life Sciences platform (GS-FLX; Roche Applied Science, Monza, Italy) using titanium chemistry as previously described.32
A pipeline was developed to reduce the 454 errors. In particular, it is known that the 454 platform introduces potential errors as single-nucleotide substitutions and small insertions and deletions (InDels).33 GsAmplicon software (Roche Diagnostics, Branford, CT) was used for clustering and automatically correcting typical 454 errors. Reads shorter than 360 nucleotides were excluded, short InDels were removed, and all correct reads were translated and then clusterized by cd-hit program.34 Amino acid sequences, aligned using muscle software, were trimmed to cover positions 5–125 (121 amino acids). After this step, ambiguous reads were manually corrected. The median number of sequences (coverage) per patient, obtained after this correction pipeline, was 6962 reads (range: 2598–14,726) (Table 1).
To measure the accuracy of UDPS, the p17MA amplicon from 1 study patient has been cloned and sequenced in parallel by UDPS and by population sequencing (Sanger method). Any difference between the 2 methods was considered to be a GS-FLX sequencing error. The mean error rate (including amino acid insertions, deletions, and substitutions) estimated with this approach was 0.04%. Considering this error rate, a sensitivity threshold of 0.4% was adopted, that is, a value exceeding by 10-fold the error rate. All variations with frequency below this threshold were not considered in the analysis. The intrapatient amino acid diversity was calculated for each patient by Phylip ProtDIST software package (using Jones–Taylor–Thornton matrix).
The mean diversity values were compared by nonparametric Mann–Whitney test. To evaluate correlations, the Spearman rank correlation coefficient was calculated; Mid-P exact test was used to compare the mutation frequency between groups. Differences with P-values <0.05 were considered statistically significant.
Diversity of p17MA
It is generally proven that proviral DNA represents a more heterogeneous viral population as compared with circulating genomes, including variants that are archived and are no longer replicating. Consistent with this concept, in this study, the frequency of p17MA variants was assessed on genomes from circulating virions. Plasma samples from 18 HIV-1–infected patients with detectable viremia were analyzed by UDPS, targeting amino acid positions 5–125 of p17MA, to obtain an accurate characterization of viral quasispecies in this region. The median amino acid diversity was 160.8 (range: 40.9–533.9) substitutions per site × 10−4, a value comparable with that observed in the highly variable genomic env-V3 region. In fact, previous observation by our group, based on UDPS, indicated that the median V3 amino acid diversity observed in patients with early or chronic infection was 128 (range: 40–3818) and 235 (range: 56–1525) amino acid substitutions per site × 10−4, respectively,32,35 in agreement with data obtained with standard sequencing from other groups.36,37 No correlation between p17MA diversity and viral load or CD4 count was observed (r = 0.318, P = 0.198 and r = 0.108, P = 0.668, respectively), in keeping with the results obtained for other viral genome regions.37,38
Recently, a p17MA NH2-terminal region, designated AT20 (9–28 amino acids), has been identified as critical for most stages of HIV-1 life cycle.23 Based on its functional relevance, we hypothesized that this region could be more conserved than the remaining parts of the protein. Therefore, the extent of diversity at the NH2-terminal portion of p17MA (residues 4–28), containing the whole AT20, and the diversity of the remaining part of the protein (residues 29–125) were calculated separately. The results, shown in Table 2, indicate that the AT20-containing part of p17MA is significantly more conserved as compared with all the rest of the viral protein.
Predicted Amino Acid Variants in p17MA Quasispecies
The occurrence of predicted amino acid variations along the entire p17MA (with respect to the reference BH10 sequence) was then analyzed, including both InDels.
In Figure 1, the distribution of insertions (panels A), deletions (panel B), and amino acid substitutions present as predominant (panel C) or minority (panel D) variants is shown. On the whole, InDels were exclusively detected at the COOH-terminal region, whereas amino acid substitutions were distributed along the entire region, without a distinctive or recurrent pattern. The complete list of deletion and insertion, with their respective frequency within each patient, is reported in Tables 3 and 4, respectively. Deletions were detected in 7 patients: 3 of them (patients 6, 9, and 10) showed a single amino acid deletion, 2 (patients 1 and 16) presented 2 or more contiguous residues deleted. Most deletions were present as minority variants, accounting for 0.47%–2.47% of the viral quasispecies, whereas only 1 deletion was observed virtually in the whole quasispecies (99.93%, in patient 9). Insertions were detected in 6 patients, with a more complex and heterogeneous pattern. In fact, in most cases (patients 7, 8, 10, and 15), multiple amino acids were inserted in variable order. In all but 1 patient, 1 insertion variant was largely predominant over the others, representing >90% of the quasispecies.
In Figure 2, the frequency of mutations observed in individual patients at each position is shown. A few positions (namely, 94 and 124) presented variants virtually in all patients, as minor or dominant components (intrapatient frequency ranging from 1.93% to 100% and from 0.45% to 99.97%, respectively), representing always conservative mutations. In some amino acid stretches (residues 20–24, 35–45, 97–102), the variants were present, if any, only as minor components. In the remaining positions, either minor or predominant variants were observed. A detailed list of amino acid substitutions detected in each individual patient, with their respective frequency, is reported in Table S1 (see Supplemental Digital Content, http://links.lww.com/QAI/A516).
HIV-1 sequence variation has been widely investigated to detect and monitor the immune pressure on the virus and to study its adaptation to the human host. The recent availability of high-throughput sequencing methods, on the whole referred to as UDPS, provided an enormous improvement in the resolution power of quasispecies analysis and allowed the detection and quantification of the relative frequency of even rare variants as compared with the dominant virus population.39–41 This approach is also important in tracing the HIV-1 evolutionary trajectory and may have strong implications for novel HIV-1 therapeutic or vaccine development strategies.
HIV-1 p17MA plays a key role in the virus life cycle and is a favorite target of both humoral and cytotoxic immune responses.23 Despite its biological relevance in HIV-1 replication and pathogenesis, few attempts have been performed to analyze the presence of p17MA quasispecies.42–44 Previous analyses were mostly focused on the extent of intrahost evolutionary rates during primary infection and showed that p17MA sequence variations usually overlapped, in any given individual, with known sites functionally relevant for viral replication or immunogenic epitopes mediating cytotoxic T-cell responses.44 In the present study, by exploiting the power of UDPS, we were able to detect and quantify p17MA variants in viral genomes present in the plasma of 18 chronically HIV-1–infected patients with plasma HIV-RNA values of at least 10,000 copies per milliliter. The intrapatient amino acid diversity ranged between 40.9 and 533.9 mean substitutions per site × 10−4, comparable with that of the highly variable genomic env-V3 region of HIV-1.32,35 Amino acid variants were scattered along the entire sequence of the viral protein.
From classical clonal sequencing approaches, the intrapatient p17MA variability ranged from about 0.5% to 5%,36,45 but could be as high as 10%.46 However, from these studies, it was not possible to obtain high resolution of viral quasispecies due to the limited number of individual sequences analyzed for each patient. In the present study, based on UDPS, we could appreciate and quantify the presence of numerous variants, eventually represented as minority components of viral quasispecies within each individual patient, indicating a high p17MA genetic variation. Previous studies have shown that higher nucleotide substitution rates in Gag are associated with increased HIV-1 RNA levels in plasma, suggesting that higher levels of HIV-1 replication result in higher intrahost nucleotide substitution rates, at least during primary infection.43 It is likely that the presence of such a p17MA heterogeneous circulating viral population in our patients may reflect their history of chronic HIV-1 infection with a relatively high viremia at the time of testing. Lack of information on duration of the infection in our clinical records hampered the possibility to confirm this hypothesis, which deserves to be investigated in future studies based on a larger series of well-characterized HIV-1–seropositive individuals.
As predicted, the NH2-terminal region of the p17MA that includes the functional AT20 domain (spanning from amino acid 9 to 28) resulted significantly more conserved as compared with the remaining portion of the viral protein. This is in line with previous data showing that the AT20 functional loop is a structural constraint being an hot spot for the interaction with several cellular proteins.14,15,30,47–49 In fact, virions carrying critical mutations in this highly basic NH2-terminal domain do harbor not only an early entry defect but also display a severe impairment in their ability to incorporate envelope.6,16 In support of the evidence of the key role of AT20 loop in the HIV-1 life cycle, we have previously shown that even in the presence of nonsynonymous mutations in the AT20 epitope, their presence is not capable of affecting the spatial conformation of the loop to an extent that the folded state is destabilized.50
Interestingly, p17MA was shown to carry different deletions and insertions, largely included within its COOH-terminus and in particular in the region comprised between amino acids 112 and 121. This region has to be considered evolutionarily relevant, as mutations in this portion of the protein may generate HIV-1 variants with unaltered fitness properties.6 Indeed, recent data point to the COOH-terminus of p17MA beyond residue 119 as partially unfolded and fully disordered.19,20 This implicates that mutations in this disordered region are expected to have little impact, if any, on protein folding and stability. Moreover, the COOH-terminal helix (H5) terminates at residues ranging from 109 to 119 in various solutions and crystal structures of p17MA.20 This suggests that residues 109–119 can be partially structured, so that mutations in this amino acid stretch can be potentially tolerated at the structural level. The absence of constraints in the p17MA COOH-terminal region, therefore, does not limit the number and location of InDels that can be accepted per replication cycle. It is worth noting that the highly dynamic nature, related to the high propensity of p17MA for intrinsic disorders in the COOH-terminal region, is a characteristic feature of HIV-1 and its closest relative simian immunodeficiency virus (SIV) and HIV-2,19 being probably linked to the successful evolutionary trajectory of these retroviruses. Indeed, recent studies have highlighted the importance of p17MA as key modulator of viral fitness following SIV transmission to the new human host.51–53 In particular, Wain et al51 identified 1 site in the p17MA (amino acid 30), which encodes a Met or Leu in SIV but switched to an Arg or Lys in many current pandemic HIV strains, as the major determinant of SIV replication fitness in humans.
The propensity of p17MA for intrinsic disorders may shed new light on the biological relevance of this protein in HIV-1 pathogenesis. Usually, different biological functions can be performed by a protein that lacks ordered and/or secondary structure.54–56 However, proteins characterized for their high propensity for intrinsic disorders are known to help virus evading detection by the immune system.19 Different investigators have hypothesized an important role of p17MA in the pathogenesis of AIDS,22,23 being it a favored target of both humoral and cytotoxic immune responses. In particular, high levels of p17MA antibodies are correlated with slower progression to AIDS.57,58 It is well established that selection pressure exerted by host immune response shapes the genetic variations of HIV-1.59 The presence of deletions and insertions in a restricted area of the p17MA COOH-terminal region, comprised between amino acid 112 and 121, may be suggestive of immune mechanisms at work with different modality in different patients. This hypothesis may, at least in part, explain the presence of insertions in only 6 of 18 patients, with a more complex and heterogeneous pattern than deletions. In this respect, it is worth noting that in all but 1 patient, 1 insertion variant was largely representing the predominant quasispecies. However, it cannot be completely ruled out that accumulation of deletions and insertions in a restricted area of the COOH-terminal region might be dependent on the severe fitness impairment caused by similar insertions in other less flexible and ordered regions of the viral protein. We could not find any correlation between the presence of deletion/insertions in the COOH-terminal portion of the p17MA and viral load, CD4 count, or exposure to antiretrovirals. However, this issue is pivotal in understanding the pathogenic implications of p17MA variability, and further studies are needed to specifically address this point.
We have recently shown that in B cells, a p17MA variant derived from a Ugandan HIV-1 strain A1, named S75X, differently from the prototype clade B isolate BH10 p17MA, increases B-cell proliferation and clonogenicity on soft agar,21 providing the first evidence on the existence of a p17MA variant with oncogenic activity on human B cells. Moreover, this study showed the role of the COOH-terminal region in modulating the p17MA signaling pathway and oncogenic activity, so highlighting the complexity and consequent implications of p17MA binding to its receptor. Considering the functional relevance of the COOH-terminus of p17MA, our results pave the way for further studies aimed at unraveling possible correlations between the presence of distinct p17MA COOH-terminus variants and peculiar clinical evolutions of HIV disease, including the possible development of HIV-related tumors.
In summary, our study is the first demonstration of a high complex p17MA quasispecies in the genomes of circulating virions from chronic HIV-1–infected patients using UDPS. In fact, we detected previously unknown p17MA variants characterized by amino acid substitutions scattered along the entire p17MA sequence, with deletions and insertions located in a restricted area of the COOH-terminal region. The results presented herein provide the rational background for further studies aimed at assessing whether a higher prevalence of distinct insertions and/or deletions in the p17MA COOH-terminus may be detected in circulating virion RNA or in proviral DNA genomes from patients in different stages of HIV-1 disease. This may lead to the identification of amino acid signatures of potential prognostic and therapeutic relevance.
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