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Differential utilization of CD4+ by transmitted/founder and chronic envelope glycoproteins in a MSM HIV-1 subtype B transmission cluster

Bouvin-Pley, Mélaniea; Leoz, Marieb; Roch, Emmanuellea; Moreau, Alaina; Migraine, Juliea; Bellini, Nicolasc; Blake, Oliviad; Mammano, Fabriziod; Braibant, Martinea; Plantier, Jean-Christopheb; Brand, Denysa

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doi: 10.1097/QAD.0000000000002690

Abstract

Introduction

An increasing proportion of new HIV-1 infections in Europe and North America are occurring in transmission clusters, particularly in the population of men who have sex with men (MSM) [1–4]. In addition to epidemiological considerations, these groups of individuals infected with closely related viruses are of particular interest for studies of the early events in mucosal HIV-1 acquisition. Only one or a limited number of viral variants, known as transmitted/founder viruses, establish infection in new hosts, regardless of the genetic diversity in the viral population present in the transmitter [5–14]. This phenomenon, known as a ‘transmission bottleneck’, occurs in most cases of transmission between MSM [15–18]. However, the mechanism underlying the selective transmission of a limited number of variants remains poorly understood [5,19,20], despite the numerous studies analyzing genotypic and phenotypic differences between transmitted/founder viruses and the viruses present in individuals with chronic infection (referred to hereafter as ‘chronic viruses’) [7,10,11,15,21]. Transmitted viruses have been shown to make preferential use of the CCR5 coreceptor and to display tropism for CD4+ T cells [5,7,10–12,14,22]. However, no other consensus has emerged concerning the traits underlying the selective transmission of particular HIV-1 variants. Several reports have suggested that transmitted subtype A, C and D transmitted/founder viruses harbor Env proteins with shorter variable loops and fewer potential N-linked glycosylation sites (PNGS) [7,23–25] than chronic viruses, but this finding has not been confirmed for subtype B viruses [15,17,26]. Other studies have reported an enhanced infectivity and/or replicative capacity of transmitted/founder viruses [21,27], but again, conflicting results have been obtained in other studies [15,28]. There is also no general agreement about the role of the innate immune system, and particularly of the type I IFN-α response, in HIV-1 transmission [15,21,28,29]. The characterization of phenotypic properties specific to transmitted/founder viruses remains challenging. The primary difficulty lies in recruiting study participants at an early stage of infection and identifying the transmitting partner. Indeed, several studies have compared transmitted and chronic viruses in the absence of information about transmission [14,21,26,30], and only a few studies have investigated the phenotypic properties of epidemiologically linked transmission pairs [8,15,28,31,32].

We compared the genetic and phenotypic properties of the Env proteins of clade B viruses from four MSM with primary infections and one chronically infected MSM from the same transmission cluster. This approach provided us with a unique opportunity to investigate whether transmitted/founder viruses had features in common that could be used to distinguish them from closely related chronic variants.

Methods

Study population

Five HIV-1-positive individuals were identified as belonging to a transmission cluster on the basis of the phylogenetic relatedness of their HIV-1 polymerase-reverse transcriptase (pol-RT) sequences. They were all MSM, infected with subtype B viruses and without antiretroviral treatment (ART) at the time of sample collection. Four study participants were sampled during primary infection (FO, MP, CO and XE) and one participant was chronically infected (PU) (refer to Table, Supplemental Digital Content 1, https://links.lww.com/QAD/B842). The date of infection was estimated on the basis of laboratory data (western blotting pattern) with acute infection defined as documented seroconversion within 90 days of the estimated date of transmission, and chronic infection defined as at least 180 days having elapsed since seroconversion [33]. Plasma/serum samples were obtained from registered collections (AC 2014-2293) of routine blood samples and the National Reference Centre for HIV at Rouen University Hospital.

Single-genome amplification of the env gene and analyses of deduced amino acid sequences

HIV-1 RNA was extracted from plasma with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with the outer primer ExtM3 (5′-CTTRTAAGTCATTGGTCTTAAAGGYAG-3′) and the Superscript III First strand synthesis system (Invitrogen, Carlsbad, California, USA). Single-genome amplification (SGA) of the full-length HIV-1 env was performed with the outer primer pair ExtM5 (5′-ATGGCAGGAAGAAGCGG-ARRC-3′) and ExtM3 and the inner primer pair IntM5XE (5′-AATTCTCGAGAATTCAGAAAGAGC-AGAAGACAGTGGCAATG-3′) and IntM3MX (5′-GGCCACGCGTCTAGACTACTTTTTGACCACT-TGCCMCCCAT-3′), as described elsewhere [9,11]. All products were sequenced directly, according to the Dye Terminator cycle sequencing protocol (Applied Biosystems, Foster City, California, USA). The env sequences obtained were deposited in GenBank under accession numbers MN690612 to MN690734.

Nucleotide sequences were aligned with CLUSTAL W and edited manually to remove ambiguous regions. PNGS were identified with the N-Glycosite tool from the HIV database website (http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html). Amino-acid positions were identified according to standard HXB2 numbering. Sequence logos were generated with the WebLogo application [34]. Evolutionary history was inferred by using the maximum likelihood method based on the General Time Reversible model [35]. Evolutionary analyses were conducted in MEGA7 [36]. The phylogenetic tree was built using the rainbow tree tool at the HIV database website (https://www.hiv.lanl.gov/content/sequence/RAINBOWTREE/rainbowtree.html). The time since the most recent common ancestor (MRCA) was estimated using the Poisson fitter tool at the HIV database website (https://www.hiv.lanl.gov/content/sequence/POISSON_FITTER/pfitter.html).

Production of pseudoviruses and analysis of their envelope glycoprotein content and infectivity

We inserted 13 env SGA products representative of the viral populations infecting each participants into the pCI expression vector (Promega, Madison, Wisconsin, USA). Env-pseudoviruses were produced, as previously described [37], by cotransfecting 5.0 × 106 293T cells with 4 μg pCI-env and 8 μg pNL4.3.LUC.R_E [38] containing either the gag sequence encoding the matrix protein of the NL4.3 strain or a gag sequence representing the viral population infecting each study participant. These gag sequence (accession numbers MN690735 to MN690739 in Genbank) were obtained by conventional sequencing of bulk PCR product and were inserted between the BssHII and SpeI sites of pNL4.3.LUC.R_E. Virus stocks were harvested after incubation of the transfected cells for 72 h. They were purified by filtration (0.45 mm pores) and stored as aliquots at −80 °C.

For the analysis of Env content, a suspension of viral particles was overlaid on a 20% sucrose cushion and subjected to centrifugation at 87 000 × g for 1.5 h at 4 °C. The resulting viral pellets were solubilized by incubation overnight at 4 °C in 100 μl PBS supplemented with 1% Triton X-100 and an antiprotease cocktail. P24 antigen content was determined by ELISA (INNOTEST HIV Antigen mAb; Innogenetics, Ghent, Belgium). Quantitative Env ELISA was performed in Nunc Maxisorp plates (Dutscher, Brumath, France), as previously described [39,40].

Viral infectivity was determined in quadruplicate in TZM-bl cells, for calculation of the median culture infectious dose 50 (TCID50), as previously described [41,42]. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by a Ficoll gradient (Histopaque; Sigma-Aldrich, St. Louis, Missouri, USA) and treated after centrifugation with 5 μg/ml of phytohemagglutinin in Roswell Park Memorial Institute medium (RPMI) 1640 medium supplemented with 10 ng/ml of IL-2 (Roche, Basel, Switzerland), 20% fetal bovine serum (FBS) and antibiotics for 3 days. They were then washed and maintained in RPMI 1640 medium supplemented with IL-2, 20% FBS and antibiotics as previously described [43,44]. Monocytes were isolated from PBMCs by negative selection using the EasySep Human Monocyte Enrichment Kit without CD16 Depletion (Stemcell Technologies, Vancouver, British Columbia, Canada). Monocyte-derived macrophages (MDM) were obtained by allowing harvested monocytes to differentiate into macrophages for 7 days in RPMI 1640 medium supplemented with 10 ng/ml human M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany). Viral infectivity was determined in quadruplicate in MDM or PBMCs. Briefly, 1.5 × 105 MDM or PBMCs were plated per well in 96-well plates the day prior to infection. Cells were infected with 100 ng of p24 virus equivalent in a final volume of 200 μl per well. Infection levels were determined after 48 h by measuring the luciferase activity of cell lysates using the Bright-Glo luciferase assay (Promega) and a Centro LB 960 luminometer (Berthold Technologies, Bad Wildbad, Germany). Wells producing relative luminescence units (RLU) more than 2.5 times the background were scored as positive.

Inhibition of virus entry by maraviroc, enfuvirtide, IFN-α, antihuman CD4+ antibodies and soluble CD4+

TZM-bl cells were used in duplicate experiments to assess the sensitivity of pseudotyped viruses to the CCR5 antagonist maraviroc (MVC) (NIH AIDS Reagent Program), the fusion inhibitor enfuvirtide (ENF) (NIH AIDS Reagent Program), the monoclonal antihuman CD4+ antibodies LM52 (NIH AIDS Reagent Program) derived from the humanized monoclonal anti-CD4+ antibody ibalizumab [45,46], and SK3 (Biolegend, San Diego, California, USA), and the CD4+ inhibitor sCD4-183 (NIH AIDS Reagent Program), as previously described [40]. Infection levels were determined after 48 h, by measuring the luciferase activities of cell lysates. IC50 values were calculated as the inhibitor concentration decreasing RLU values by 50%. Results are expressed as mean values.

The sensitivity of pseudotyped virus (1000 TCID50 per well) to LM52 (1 μg/ml) or sCD4-183 (50 μg/ml) was also evaluated in MDM by measuring the luciferase activities of cell lysates (RLU), as described above.

IFN-α sensitivity was assayed in MT4-R5 cells [47], with a range of concentrations of IFN-α-2b (from 3.2 to 104 pg/ml), as previously described [48]. The IC50 was calculated as described above.

Statistical analysis

The statistical significance of differences between the medians of transmitted/founder variants and chronic variants was determined in Mann–Whitney tests. Correlations between continuous variables were assessed with Spearman's rank correlation test.

Results

Study population

We selected the five individuals for study on the basis of the phylogenetic linkage of their HIV-1 pol-RT DNA sequences following routine ART resistance testing (Fig. 1a). Four of the five members of this cluster were acutely infected individuals with a high viral load (median value of 6.8 log10 copies/ml), typical of the early stage of infection (refer to Table 1, Supplemental Digital Content 1, https://links.lww.com/QAD/B842). Three were at Fiebig stage IV (FO, MP, XE) and the fourth was at Fiebig stage VI (CO). The fifth patient (PU) tested HIV-1-positive with no information about the duration of infection and was considered to be chronically infected, having reached a viral set point of 4.0 log10 copies/ml, a complete western blotting pattern, and showing no clinical signs of acute infection.

F1
Fig. 1:
Phylogenetic analyses of HIV-1 pr-RT, full-length Env sequences and comparison of variable-loop length and potential N-linked glycosylation sites numbers.

Comparison of envelope glycoprotein amino-acid sequences

In total, 123 SGA-derived full-length Env sequences were obtained from the five individuals, with a range of 20–30 sequences per study participant (Fig. 1b and Supplemental Digital Content 1, https://links.lww.com/QAD/B842). Evolutionary history was inferred by the maximum likelihood method (Fig. 1b). Mean genetic diversity was low among the study participants with acute infection, ranging from 0.001 to 0.004, whereas the viral population of the chronically infected individual (PU) displayed broader genetic diversity, as expected (P distance = 0.031), confirming the occurrence of a genetic bottleneck during transmission. Based on previous reports relating HIV-1 sequence evolution of 1% increase per year [49,50], the mean diversity of the sequences from the five subjects could discriminate recent from chronic infections. Moreover, two transmitted/founder viruses appeared to have initiated the infection in the study participant FO, founding different genetic lineages. The sequences sampled from subject XE were characterized by a mean genetic diversity identical to that in FO (0.004), but without clearly identifiable distinct lineages. A single transmitted/founder virus was identified for each of the remaining acutely infected study participants, CO and MP. The time since the MRCA was estimated using the Poisson fitter tool available on HIV LANL database tool. The estimated age of the viral population ranged from 12 days for MP to 78 days for FO (refer to Table 1, Supplemental Digital Content 1, https://links.lww.com/QAD/B842). The estimated time was higher for XE and FO due to a heterogeneous infection for these subjects [51]. The Env sequences of these transmitted/founder variants were interspersed with those of the chronically infected participant PU, confirming the relatedness of these viral sequences. Nevertheless, no identical Env amino-acid sequence common to two different study participants, or transmitted/founder variants closely related to the chronic variants were found. To identify if the viral population of PU could result from a superinfection event, the Env sequences of the viruses included in our study were aligned with 59 Env sequences isolated from French clade B chronically infected study participants (refer to Phylogenetic analysis, Supplemental Digital Content 2, https://links.lww.com/QAD/B843). The absence of a distinct viral population from subject PU, branching outside the cluster of sequences from the five patients studied here on the rainbow tree, suggested a single transmission event.

Env sequences derived from transmitted/founder variants have been reported to harbor shorter gp120 variable regions and fewer PNGS than those from chronic variants [7,23,24,52–54]. With a few exceptions (e.g. some MP variants had a shorter V1 region and some XE variants had a longer V4 region; Fig. 1c and e), the variable regions were highly conserved in terms of both their length and the number of PNGS between transmitted/founder and chronic variants (Fig. 1d, e, g and h). Similarly, none of the specific signature sites previously described in transmitted/founder variants, such as the H12, T283, N362, Q389, E429, T465 and G471 residues, could differentiate between Env sequences derived from our transmitted/founder and chronic variants [18,55,56]. A detailed comparison of amino-acid sequences also found no consistent differences in residues important for maintaining the gp120-gp41 interface, involved in coreceptor [57] and receptor binding [58] or in the kinetics of viral entry [59–61].

Transmitted/founder and chronic variants have similar infectious properties

We analyzed the infectious properties of 13 pseudotyped HIV-1NL4-3 bearing envelope glycoproteins representative of the main genetic lineages previously identified in the participant virus populations (i.e. FO E21 and E54, XE B6 and B14, CO 149, MP H14, PU 11, 17, 30, 49, 55, 235 and 239; Fig. 1b). Infectivity varied between pseudoviruses, with a relative SD (RSD) more than 17% and means of log10 TCID50/25 ng of p24 ranging from 1.98 for XE B14 to 4.38 for MP H14 (Fig. 2a). However, as illustrated in Fig. 2b, the median infectivity of the transmitted/founder variants (7.6 × 103 TCID50/25 ng of p24) was similar to that of chronic variants (1.1 × 104 TCID50/25 ng of p24, P = 0.80 in a Mann–Whitney test).

F2
Fig. 2:
Comparison of infectivity and Env contents between pseudotyped HIV-1NL4-3 bearing transmitted/founder and chronic Env.

The NL4–3 background used to generate the pseudoviruses may have modified Env incorporation by altering the interplay between the cytoplasmic tail of gp41 and the matrix domain of Pr55gag during viral assembly. We therefore analyzed the gp120 content of each virus by ELISA (Fig. 2c). We found that gp120 levels differed considerably between the various pseudoviruses (gp120/p24 ratio ranging from 0.063 for XE B14 to 0.636 for XE B6, with a RSD > 58%). Taking the Env content of each variant into account, we then assessed infectivity normalized for Env content (1 ng) by dividing the TCID50 values by the gp120/p24 ratios (Fig. 2d). The variants differed in infectivity, but the median values of transmitted and chronic variants were similar (716.4 and 1.4 × 103 TCID50/ng of gp120, respectively, P = 0.23 in a Mann–Whitney test; Fig. 2e). These data suggest that HIV-1NL4-3 bearing Env derived from transmitted/founder or chronic variants have similar infectious properties in this transmission cluster. A similar trend was observed in primary PBMCs (Fig. 2f). The median values of infectivity (RLU), using 100 ng of p24 virus equivalent for each transmitted/founder and chronic variant tested, were similar (1.4 × 104 and 1.2 × 104 RLU/100 ng of p24, respectively, P = 0.37 in a Mann–Whitney test; Fig. 2f). Therefore, no difference in infectivity was observed in cells known to express high levels of CD4+ with HIV-1NL4-3 bearing Env derived from transmitted/founder or chronic variants.

Given that TZM-bl cells and PBMCs express high levels of CD4+[44], we further explored whether a difference in infectivity between transmitted/founder and chronic variants could be observed in macrophages known to express a lower density of CD4+[62]. MDM were infected in a single round of infection by transmitted/founder and chronic pseudoviruses and infectivity level was evaluated 48 h postinfection by measuring the luciferase activity (RLU). Similarly to what we observed in TZM-bl cells and PBMCs, there is no difference in infectivity between transmitted/founder and chronic variants (Mann–Whitney test, P = 0.84) (Fig. 2g). Thus, the infectivity of viruses issued from this transmission cluster appears similar in both the TZM-bl cells line and the primary cells.

We then investigated whether HIV pseudovirus infectivity was affected by an impairment of the interaction between the cytoplasmic tail of gp41 and the matrix domain of NL4-3 Pr55gag, by replacing the gag sequence of pNL4.3.LUC.R_E with the corresponding sequence representative of the viral population infecting each study participant, to obtain Env-MA-pseudoviruses, as described above (Fig. 3a). Env incorporation levels were higher with the Env-MA-pseudotyped viruses (Fig. 3b and d), but the infectivity of these viruses was lower than that of pseudoviruses harboring the NL4.3 matrix (Fig. 3c). Nevertheless, no difference in infectivity was observed between acute (obtained from a study participant with acute infection) and chronic viruses (median values of 174.2 and 82.8 TCID50/ng of gp120, respectively, P = 0.73 in a Mann–Whitney test; Fig. 3e). These results confirmed that the viral variants derived from the acutely infected study participants were not more infectious than those from the chronically infected study participant.

F3
Fig. 3:
Comparison of infectivity and Env contents between pseudoviruses harboring the NL4.3 or the study participants’ matrix (MA) proteins.

Transmitted/founder and chronic variants have similar susceptibilities to maraviroc and enfuvirtide

Several studies have reported that the transmitted viruses seem to be more restricted in the use of CCR5 coreceptor, as revealed by their greater sensitivity to the CCR5 antagonist MVC [7,30]. We first verified the R5 tropism of our 13 pseudoviruses by measuring their ability to infect exclusively the CCR5+/(CD4+)/U373 cells [63]. We then assessed the efficiency of pseudovirus to use CCR5 by infecting TZM-bl cells in the presence of various concentrations of MVC (6000 to 0.3 nmol/l). We observed that all pseudoviruses were highly susceptible to MVC, with an IC50 less than 17 nmol/l and maximal percentage inhibition (MPI) values above 99% in the presence of saturating concentrations of MVC (Fig. 4a and b). We found no significant difference in median MVC IC50 between the transmitted/founder (6.42 nmol/l) and chronic (4.96 nmol/l) variants (P = 0.19 in a Mann–Whitney test; Fig. 4c), suggesting that CCR5 usage in the TZM-bl cell model, explored through sensitivity to MVC, is not one of the main determinants of the selective process observed during HIV transmission.

F4
Fig. 4:
Sensitivity to the CCR5 antagonist maraviroc, the fusion inhibitor enfuvirtide and IFN-α 2b.

We also tested the susceptibility to the fusion inhibitor ENF, which mimics the sequence of the HR2 domain of gp41 and binds to the HR1 domain exposed after CD4+/coreceptor engagement [64]. As for MVC, we observed no difference in the sensitivity to ENF of Env proteins derived from transmitted/founder and chronic variants (median IC50 of 0.08 versus 0.06 μg/ml; P = 0.71), suggesting that the kinetics of viral entry were similar for all pseudoviruses (Fig. 4d).

Sensitivity to IFN-α does not differ significantly between transmitted/founder and chronic variants

Type 1 interferons control HIV replication [65]. We investigated whether the Env proteins derived from the transmitted/founder viruses studied here could induce lower susceptibility to IFN-α than the Env proteins from chronic variants, by measuring the efficiency of viral infection in MT4-R5 cells in the presence of increasing concentrations of IFN-α [48]. IFN-α sensitivity did not differ significantly between the transmitted/founder variants and the chronic variants (median IC50 of 7.60 versus 8.03 pg/ml; P = 0.36) (Fig. 4e). Similarly, transmitted/founder and chronic variants had similar MPI values at a concentration of 2 ng/ml IFN-α (not shown). Of note, we used pseudotyped viruses competent for a single round of infection, allowing to explore the effects of IFN-α only on the steps between virus adhesion and protein production.

Transmitted/founder variants are less susceptible to soluble CD4+

We investigated viral entry further, by comparing the CD4+ receptor usage of our 13 pseudoviruses based on their sensitivities to the soluble form of CD4+ (sCD4-183). The pseudoviruses differed considerably in sensitivity to sCD4-183, with a RSD more than 84% and mean IC50 ranging from 4.35 for PU 17–44.5 for XE B14 (Fig. 5a). In contrast to our previous findings for other entry inhibitors, the median IC50 of sCD4-183 was significantly higher in the transmitted/founder variant group than in the chronic variant group (15.18 versus 6.98 μg/ml; P = 0.01; Fig. 5b). Significantly, the transmitted/founder variants also displayed enhanced sensitivity to occupation of the CD4+ receptor by the anti-CD4+ mAbs LM52 (median IC50 of 0.038 versus 0.056 μg/ml, P = 0.05) and SK3 (median IC50 of 0.037 versus 0.048 μg/ml, P = 0.01) (Fig. 5c and d). Lower susceptibility to CD4+-mimetics and higher susceptibility to antibodies that occupy CD4+ on target cells are indicative of reduced capacity to interact with CD4+[66]. Significant inverse correlations were found between the LM52 and sCD4 IC50 values (Spearman r = −0.692, P = 0.01) and between the SK3 and sCD4 IC50 values (Spearman r = −0.637, P = 0.02), suggesting that Env glycoproteins from transmitted/founder variants bind less efficiently to CD4+ than those of chronic variants (Fig. 5e and f).

F5
Fig. 5:
Sensitivity to the CD4+ receptor inhibitors sCD4-183, LM52 and SK3.

We further checked whether the differential sensitivity to inhibitors targeting the interaction between Env and CD4+ could also be observed in cells expressing low levels of CD4+ receptors, that is MDMs. For this purpose, we analyzed the sensitivity of the pseudoviruses normalized to 1000 TCID50/ml in the presence of a maximum inhibitory concentration of sCD4-183 (50 μg/ml) or LM52 (1 μg/ml). Consistent with our findings in TZM-bl cells, we found that transmitted/founder variants are less sensitive to sCD4-183 (median RLU/1000 TCID50 of 2.0 × 104 versus 5.6 × 103, P = 0.07) and more sensitive to LM52 (median RLU/1000 TCID50 of 1.9 × 102 versus 1.2 × 103, P = 0.05) (Fig. 5g). Overall, the analysis of the sensitivity to the CD4+ inhibitors revealed subtle differences in CD4+ receptor utilization between transmitted/founder viruses and chronic viruses. We therefore investigated a 51-amino acid region encompassing the CD4+ binding loop between Env amino acids 338–389 in transmitted/founder and chronic variants. Amino-acid variants were identified at positions 339, 342, 346, 349, 352, 353, 356, 357, 362, 363, 364 and 389 (Fig. 5h). This variability could explain the difference in sensitivity to soluble CD4+, as recently described [67–69]. None of the previously described specific molecular determinants, such as the R373E, S375W, N377V or G380P substitutions, were identified [68,70], but the combination of mutations found here and around the CD4+ site may be responsible for these differences.

Discussion

We investigated the genetic bottleneck occurring during HIV-1 transmission by studying the genetic and phenotypic properties of Env variants derived from four study participants identified during primary infection and one chronically infected individual, all from the same transmission cluster. We hypothesized that a comparison of genetically linked transmitted/founder and chronic HIV-1 variants would facilitate the identification of common features crucial for the earliest stages of HIV-1 infection. Given the key role of Env in viral entry, we focused on Env-mediated viral properties relating to the entry of the virus into its target cell.

The SGA of env genes, sequencing, and phylogenetic analysis confirmed the close relationship between the viral variants derived from the individuals within the transmission cluster and the occurrence of a genetic bottleneck upon transmission. However, a detailed comparison of deduced Env amino-acid sequences showed no difference in the length of variable loops, number of glycosylation sites and known signature residues between the acute and chronic variants, suggesting that these sequence features are not determinants of the selection of variants during the transmission events investigated here.

We further explored the differences between transmitted/founder and chronic variants by studying their functional properties using 13 pseudoviruses expressing SGA-derived Env variants representative of different genetic lineages found in the individuals’ virus populations. Infectivity was similar for all the Env variants, regardless of whether the normalized input was based on p24 and Env content, both in cells expressing a high level of CD4+ receptors and in macrophages or whether the pseudoviruses harbored homologous Env and matrix sequences. We considered other phenotypic properties of transmitted/founder viruses, by also investigating sensitivity to various entry inhibitors, including the CCR5 antagonist MVC, the fusion inhibitor ENF, a CD4+ analog, two anti-CD4+ antibodies, (LM52 and SK3) and IFN-α. Susceptibilities to MVC and ENF were within the same range, for all pseudoviruses. Similarly, no trend towards IFN-α resistance was found in transmitted viruses. However, as our study focused on the impact of HIV-1 envelope glycoproteins, any effect of IFN-α on late steps of viral replication would not have been detected.

By determining sensitivity to soluble CD4+ (sCD4-183) as a surrogate for the efficiency of CD4+ interaction [71,72], we showed that the major property distinguishing our transmitted/founder variants from the chronic variants within this cluster of transmission was their greater resistance to soluble CD4+. These results suggest that transmitted/founder variants have a lower affinity for CD4+ and that they may require higher levels of CD4+ for the efficient infection of target cells. CD4+ is expressed at high density on CD4+ T cells, but at a much lower density on other CD4+-expressing cells, such as monocytes or macrophages [73]. Consistent with our data, several studies have shown that most transmitted/founder viruses infect macrophages and other cells with low CD4+ densities at low efficiency [12,22,73,74]. However, no difference in infectivity was found in MDM between transmitted/founder and chronic variants from this cluster of transmission. We assume that these data suggest that the use of CD4+ inhibitors could highlight subtle phenotypic differences, hardly detectable in primary cells. Significantly, the transmitted/founder variants also displayed enhanced sensitivity to occupation of the receptor CD4+ by the anti-CD4+ antibodies LM52 and SK3. A significant inverse correlation was found between LM52 or SK3 and sCD4 IC50 values, such that transmitted/founder variants would bind less efficiently to cells with low levels of CD4+ expression. Consistent with our findings, resistance to ibalizumab or LM52 has been shown to be associated with enhanced sensitivity to sCD4 neutralization [75]. This difference in susceptibility to ibalizumab or LM52 was mediated principally by the absence of PNGS in the V5 loop [46,75], but no difference was found between transmitted/founder and chronic variants from the transmission cluster. We further explore this differential utilization of CD4+ receptor in MDM that express low levels of CD4+ by comparing the sensitivity to sCD4-183 and LM52 between transmitted/founder and chronic variants. A similar trend is observed, suggesting that when we decrease the number of accessible CD4+ receptors to a very low level, by combining the use of the macrophages with the CD4+ inhibitors, we succeed in observing a difference in CD4+ receptor dependence between transmitted and chronic viruses while this property is not observable in the MDM only.

Given that Env affinity for CD4+ can be increased by gp120 substitutions [69,71], we compared the Env sequences of the various pseudoviruses. Surprisingly, no evident amino-acid pattern was associated with sCD4 sensitivity. Duenas-Decamp et al. showed that a small number of substitutions in the CD4+ binding loop and flanks could affect CD4+ interactions [66,68]. We therefore investigated a 51-amino acid region encompassing the CD4+ binding loop between amino-acids 338 and 389. Differences between transmitted/founder and chronic sequences potentially capable of modulating the efficiency of Env/CD4+ interactions were found at 12 positions. Nevertheless, no specific signatures focused on amino acids flanking the CD4+ binding loop was identified, as previously described [66–70]. Our results suggest a role for an association of mutations underlying differences in CD4+ receptor utilization rather than a clearly defined molecular signature. The differences in the conservation of amino acids at twelve positions between transmitted/founder viruses and chronic viruses are adjacent to the highly conserved SGGD-E CD4+ contact residues. Viruses from the cluster differ by combinations of mutations, and not by changes at key residues, that may contribute to an overall perturbation of Env-CD4+ affinity.

In conclusion, our results confirm the existence of the expected genetic bottleneck after HIV transmission, with a limited number of variants identified in the four study participants with acute infection. Most of the properties studied were common to the transmitted/founder and chronic variants, but these two groups of variants differed in their sensitivity to CD4+ receptor inhibitors, including a soluble form of CD4+ and two antibodies targeting CD4+. Our study thus provides support for the hypothesis that the lower efficiency of envelope glycoprotein binding to CD4+ in transmitted/founder variants may confer a selective advantage during the early steps of HIV transmission.

Acknowledgements

The current work was supported by Sidaction (France) and the ANRS (Agence Nationale de Recherche sur le Sida et les Hépatites Virales). M.B.-P., M.L., J.-C.P and D.B. conceived and designed the experiments. M.B-P., M.L., E.R., A.M., J.M., N.B., O.B. and F.M. performed the experiments. M.B.-P., M.L., J.-C.P., F.M. and D.B. analyzed the data and wrote the article. M.L. and J.-C.P. selected and characterized patient samples. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4.3.LUC.R-E- from N. Landau; TZM-bl cells from J. C. Kappes, X. Wu, and Tranzyme Inc.; Maraviroc (Cat #11580); sCD4-183 from Pharmacia Inc.

Conflicts of interest

There are no conflicts interest.

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Keywords:

CD4+ binding; envelope glycoproteins; genetic bottleneck; HIV transmission cluster

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