While deletions cannot be incorporated in phylogenetic analysis at present, we could examine V3-316T selection. We generated a phylogenetic tree based on subtype-C sequences from 1985 to 2005 and superimposed the nature of the amino acid at position 316 (Fig. 2a, Supplementary Fig. S2). The earliest sequences are from the Democratic Republic of Congo (DRC) where HIV is thought to have evolved and diversified. The phylogenetic tree demonstrates that both 316A and 316T could be found in the DRC sequences at the base of the tree. However, the deep lineages and branches of the tree mostly carry 316T, suggesting that this residue has been generally advantageous to subtype-C spread. Whereas 316V can be found sporadically, alanine seems to spread locally, especially in more recent sequences from South Africa and Botswana. We studied the distribution of 316A and 316T in larger alignments of shorter envelope sequences dating from 1990 to 2005 to see if this observation could be substantiated (Fig. 2b). We found that the frequency of 316T indeed had decreased from around 80% in 1990 to approximately 68% in 2005. This suggests that there may be alternate selective advantages for 316A in some situations in vivo and/or that other changes sometimes are sufficient to favor transmission of subtype C.
To analyze the prevalence of 316T in blood and cervix, we obtained samples from 36 additional women infected by HIV subtypes A (n = 3), B (n = 28) or C (n = 5). Whereas single-copy envelope sequences could be amplified from PBMC from all women, no HIV was found in the cervices of four .
Most subtype-B infected women carried virus with V3-316A, but four had either 316T, 316T/M, 316S or 316V virus in both samples (Fig. 3a). Moreover, we found V3-316 compartmentalization between blood and cervix in one subtype-B-infected woman (1/28, 4%); all genital viruses carried 316T (5/5), whereas only 316A could be found in PBMC (7/7) (Fisher's exact test, P < 0.001; Fig. 3b).
Of the five subtype-C-infected women, only one carried HIV-316A, and no virus could be amplified from her genital sample; the rest had either 316T or 316T/M in both PBMC and cervix. In one of these patients with HIV-316T, we were able to analyze gp160 from vagina as well as cervix and PBMC (Fig. 3c). Although 28% of the cervical sequences had very short V1–V2 loops (55 amino acids), such short loops were not found in blood; most vaginal sequences had intermediate V1–V2 loops. Overall, V1–V2 loop lengths differed significantly between genitalia and blood when grouped into either short (≤69 amino acids) or long (>69 amino acids) loops (Fisher's exact test, P = 0.02).
While our results demonstrate that subtype-A and subtype-B virus with 316A can replicate in the cervix, they also highlight that selective forces in the genital tract may favor replication of virus with 316T even in subtype-B-infected women. This is reminiscent of the preferential cervical replication of subtype-C virus in our dually infected woman. Furthermore, we demonstrate that in a woman carrying only HIV-316T, selective forces in the genital tract may favor shorter V1–V2 loops than selection in blood.
Subtype C is responsible for an ever-increasing fraction of new HIV infections worldwide, especially in areas where heterosexual transmission predominates, and it now prevails even in regions where subtype A or D or both used to be common [1,2,39,40]. This suggests that there are biological factors favoring transmission of subtype C, but no plausible mechanism has been suggested.
Our study of infectious, chimeric HIV clones carrying V1–V3 sequences from a woman dually infected with subtypes A and C, but with preferential cervical subtype-C replication, combined with the analysis of HIV sequences from blood and cervix from 36 additional women, lead to four findings suggesting that subtype C generally has a relative replication advantage compared with most subtype-A and subtype-B viruses in the female genital tract.
First, we identify a V3-envelope signature sequence (316T) at high frequency in subtype C, which might strengthen the interaction between HIV and CCR5, the main coreceptor in the female genital tract [41–45]. We find that the effect of 316T may be independent of the surrounding genomic context and show the effect of an A316T substitution in subtype A. Second, we find that selection for 316T can occur even in the cervix of a minority of untreated subtype-B-infected women. Third, we demonstrate that a subtype-A V1-deletion augments virus production in cell lines expressing low levels of CD4 or CCR5 and propose that the shortening of the V1 loop increases viral interactions with the CD4 receptor. Fourth, we find that genital selection in a subtype-C-infected woman favors outgrowth of very short V1–V2 loops, despite subtype C already having the most compact V1–V2 loops of all HIV subtypes.
We found that subtype C had a greater ability to replicate in PBMC cultures than subtype A and mediated more extensive virus-induced fusion of cells expressing low CCR5 levels. However, we observed marked changes in subtype-A replication patterns over time and identified adaptations in either the V1 (T114I, V1-Δ5) or V3 (N302K, A316T) region, which are known to interact with the CD4 and CCR5 receptors, respectively. These adaptations enhanced virus replication and fusion comparable to what we found using the wild-type subtype-C clones. Whereas two were found rarely, if at all, in HIV sequences obtained from infected persons, two others (A316T and V1-Δ5) resembled sequence motifs in the subtype C sequences from our patient and in most subtype C sequences from natural infections. In other subtypes, and in most CRFs, alanine is preferred at position 316, with the notable exception of CRF_02_AG, which carries threonine. Analogous to subtype-C spread in southern and eastern Africa, CRF_02_AG has spread throughout western Africa, primarily via heterosexual contact [1,46].
Both subtype-A and subtype-C chimeric viruses interacted efficiently with the CD4 receptor; however, the V1-Δ5 adaptation augmented this interaction and increased infectivity several-fold in both cell lines and PBMC. The CD4-binding region is predicted to be shielded by the V1–V2 loop and shorter loops have been associated with increased susceptibility to antibody neutralization [13–15]. As short V1–V4 loops and more neutralization-sensitive HIV variants have been found in early subtype-A and subtype-C infections, it was suggested that these were either preferentially transmitted or rapidly selected in the recipient [12–15]. We demonstrate that genital selection pressures in subtype-C-infected women may sometimes favor virus with more compact V1–V2 loops than what is commonly found in blood. Consequently, whether transmission of such variants is preferential or at times simply occurs by chance, remains to be elucidated.
Structural modeling suggested that the A316T effect might be independent of the surrounding genomic background, as a new hydrogen bond may form between the polar threonine and CCR5. The importance of position 316 in gp120-CCR5 interactions is indicated by the induction of drug-resistance mutations at this site by two different CCR5 inhibitors (A316T, maraviroc; A316V, AD101) [47,48]. CCR5-binding studies [47,48] have demonstrated that these polar and nonpolar substitutions crucially affect HIV-CCR5 interactions through different mechanisms.
Our phylogenetic analysis demonstrated that 316T must have been favorable for subtype-C transmission. However, it also revealed that 316A seems to spread locally, especially in recent sequences from South Africa and Botswana. This implies that viruses with 316T incur a viral fitness cost even in subtype C, as has been directly demonstrated for maraviroc-resistant subtype-B virus with 316T in in-vitro replication assays . Additionally, in some situations in vivo, there could be selective advantages associated with 316A and/or other changes resulting in effective transmission. The A316T substitution could also affect virus susceptibility to host antibodies or cytotoxic T cell (CTL) responses, as the V3 crown is a prime target for neutralizing antibodies and encompasses CTL epitopes restricted by at least five different human leukocyte antigen (HLA) alleles.
Whereas HIV carrying 316A is fully replication competent and can be found in cervix, virus with 316T may sometimes have a relative replication advantage as CCR5 expression has been found on cells sampled from vagina, ectocervix and endocervix [41–45,49], the proportion of cervicovaginal T cells expressing CCR5 is markedly expanded as compared with peripheral blood , HIV primarily propagates in vagina by T-cell infection  and cell-to-cell transfer is the predominant mode of HIV spread . This relative replication advantage may be greatest in women with low genital CCR5-expression levels such as premenopausal women with normal genital mucosa, and less in women with high expression levels due to, for instance, sexually transmitted diseases (STD), cellular infiltrates, menopause or oral contraceptive use [42,52]. Indeed, our patients with preferential cervical replication of HIV subtype C or B with 316T had normal genital epithelium and no STD .
We propose that the efficient HIV-316T-CCR5 interaction may increase viral transmissibility. That a polymorphism at a single amino acid site can have such profound consequences agrees with the first report of species-specific selection in primate lentiviruses, which demonstrates that efficient spread of SIV cpz in human cells depends on a single amino acid change in Gag . Our proposal is indirectly supported by transmission studies in discordant couples with known CCR5 genotype; if the uninfected partner was heterozygous for the Δ32-CCR5 allele and thus had lower levels of CCR5 on T cells and monocytes , HIV transmission decreased during both heterosexual  and homosexual [43,44] contact, although one study found no effect .
Our results raise the possibility of subtype-specific differences in the efficacy of topical genital CCR5 inhibitors and highlight the need for clinical trials on patients infected with different subtypes. Furthermore, they suggest that the maraviroc-induced drug mutation A316T could result in viruses that are more easily transmitted through sexual contact.
In conclusion, our study suggests that subtype C has evolved in ways compatible with increased sexual transmissibility. Although other envelope regions, additional viral genes and long terminal repeat promotor differences  in combination with epidemiological dynamics, such as founder effects, also must be important factors, these subtype-C motifs may contribute to its current dominance in southern and eastern Africa, which facilitated subsequent worldwide spread.
We thank the patients, Andrew Rambaut, Kim Hasenkrug, Rachel Lacasse, Angela Vincent and Nick Willcox for helpful comments; Anita Mora for graphics, Kevin Braughton and Ron Messer for technical assistance.
A.K.N.I. initiated and designed the overall study; A.K.N.I. and B.C. planned and supervised different parts of the project; A.K.N.I., B.C. and B.L.W. analyzed data and wrote the article; B.L.W., A.E.A. and A.K.N.I. performed experiments; S.C.G., E.Y.J. and D.I.S. performed the structural modeling and RONN analyses; T.d.O. and A.K.N.I. performed the phylogenetic analysis; A.K.N.I. and P.S. provided patients, clinical data and reagents; and A.J.M. contributed reagents. All authors discussed the results and approved the final version of the article.
Funding was provided by the Danish AIDS Foundation, the Nuffield Dominions Trust, Cancer Research UK, the EU (LSHG-CT-2006-031220) and MRC, UK. The authors have no competing financial interests. Supplementary documentation can be obtained on request.
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