Functional characteristics of HIV-1 subtype C compatible with increased heterosexual transmissibility

Abstract

Background: Despite the existence of over 50 subtypes and circulating recombinant forms of HIV-1, subtype C dominates the heterosexual pandemic causing approximately 56% of all infections.

Objective: To evaluate whether viral genetic factors may contribute to the observed subtype-C predominance.

Methods: Chimeric viruses were generated using V1-V3 envelope fragments from a subtype-A/C dually infected woman with preferential genital replication of subtype C. Viral adaptation, spread and cell fusion ability were evaluated in vitro using peripheral blood mononuclear cells and HeLa-CD4-CCR5 cell lines, sequencing and cloning. Structural modeling was performed using a crystal structure of gp120-CD4-X5. Phylogenetic analysis was done using subtype-A, subtype-B and subtype-C sequences from blood and cervix of 37 infected women and database sequences.

Results: We identified two envelope motifs, compact V1-V2 loops and V3-316T, which are found at high frequency throughout subtype-C evolution and affect gp120 interactions with CD4 and CCR5, respectively. When a V1-Delta5 deletion or V3-A316T was incorporated into subtype A, each increased viral fusion and spread several fold in peripheral blood mononuclear cell and cell lines with low CCR5 expression. Structural modeling suggested the formation of an additional hydrogen bond between V3 and CCR5. Moreover, we found preferential selection of HIV with 316T and/or extremely short V1-V2 loops in cervices of three women infected with subtypes A/C, B or C.

Conclusion: As CD4-CCR5-T cells are key targets for genital HIV infection and cervical selection can favor compact V1-V2 loops and 316T, which increase viral infectivity, we propose that these conserved subtype-C motifs may contribute to transmission and spread of this subtype.

Figure 1

A. Amino acid alignment of the V1-V2 and V3 region of subtypes-A, -C and the PBMC-adapted A1-A316T, A1-N302K, A2-T144I and A20-V1Δ5 viruses numbered according to HIV(HXB2). Patient-derived viral sequences had constant V1-V2 loop lengths (A=68, C=58) and the V3-loop charge within and between subtypes was identical (+4). The Δ5 deletion in A20, 144I in A2, 302K in A1 and the 316T residue in A1 and subtype-C are boxed. As HXB2 has a unique 2-amino acid insertion at position 309, the following amino acid is number 312. B. The influence of patient subtype-A and -C V1-V3 envelope sequences on PBMC infection and spread. Live viral output was measured as focus forming units per ml (FFU/ml) over time using chimeric subtype-A, -C and subtype-B control viruses (49-5 and 81A-4) at an MOI of 0.02. Results are representative of six independent infections. C. Live viral output, measured as FFU/ml, over time after PBMC infection at an MOI of 0.02 using chimeric A, PBMC-adapted A and subtype C66 virus. The PBMC donor differs from the one used in B. Results are representative of six independent infections. D. Virus titration from supernatants of infected PBMC on JC37 cells followed by p24 antibody staining. Arrows signify single foci consisting of 1-2 nuclei. E. Fusion experiment. 293T cells (CD4-, CCR5-) were transfected with either subtype-A or -C chimeric proviral DNA, overlaid with either JC37 or RC49 cells and stained using p24 antibodies. The HIV foci sizes are representative of general differences between the subtypes. F. As in d), but using A1-A316T, A1-N302K, A2-T146I and A20-V1Δ5 and JC37 cells.

Figure 2

A. Phylogenetic tree of a 219 base pair env region (HXB2 7050-7271) from 163 subtype-C sequences (1985-2005), including our patient sequences. The scale bar shows the branch length equal to 5 nucleotide changes per 100 bases (0.05). Sequences carrying 316 A and T are blue and red, respectively, branches where all sequences have the same amino acid at position 316 are collapsed and shown as colored triangles (uncollapsed tree with GenBank accession numbers, supplementary documentation, Figure 2). Ancestral branches for India and South America are labeled with * and **, respectively. AR = Argentina, BR= Brazil, BW= Botswana, DRC= Democratic Republic of Congo, ET= Ethiopia, IL = Israel, IN= India, KE = Kenya, SE=Sweden, TZ = Tanzania, UG=Uganda, UY = Uruguay, ZA= South Africa. B. Graphs show the prevalence of 316A and T in subtype A (blue) and C (red), respectively, from 1990 to 2005 (number of sequences, subtype A/C: 1985-90, n= 82/261; 1991-95, n=147/356; 1996-2000, n= 614/1519; 2001-2005, n=166/993). C. Cladogram of 263 V3 env sequences showing the distribution of 316A (blue) and T (red) in all subgroup M subtypes (M = subgroup M root).

Figure 3

A. The patient's HIV subtype and the nature of the amino acid in position 316 in PBMC (PB) and cervix (Cx) is listed; A= Alanine, M= Methionine, S-= Serine, T= Threonine, and V= Valine (n=38, as the dually-infected woman is shown as both subtype-A and -C). B. Phylogenetic tree demonstrating genital selection of 316T (red circle) and blood selection of 316A (blue circle) in a subtype-B infected woman. Bootstrap estimates above 60 are shown. Statistically significant values are generally considered to be ≥70, although no established significance threshold exist. C. Percentage of different V1-V2 loop lengths in gp160 sequences from vagina, cervix and blood from a subtype-C infected woman with V3-316T.