Origin and biology of simian immunodeficiency virus in wild-living western gorillas

Abstract

Western lowland gorillas (Gorilla gorilla gorilla) are infected with a simian immunodeficiency virus (SIVgor) that is closely related to chimpanzee and human immunodeficiency viruses (SIVcpz and HIV-1, respectively) in west central Africa. Although existing data suggest a chimpanzee origin for SIVgor, a paucity of available sequences has precluded definitive conclusions. Here, we report the molecular characterization of one partial (BQ664) and three full-length (CP684, CP2135, and CP2139) SIVgor genomes amplified from fecal RNAs of wild-living gorillas at two field sites in Cameroon. Phylogenetic analyses showed that all SIVgor strains clustered together, forming a monophyletic lineage throughout their genomes. Interestingly, the closest relatives of SIVgor were not SIVcpzPtt strains from west central African chimpanzees (Pan troglodytes troglodytes) but human viruses belonging to HIV-1 group O. In trees derived from most genomic regions, SIVgor and HIV-1 group O formed a sister clade to the SIVcpzPtt lineage. However, in a tree derived from 5' pol sequences ( approximately 900 bp), SIVgor and HIV-1 group O fell within the SIVcpzPtt radiation. The latter was due to two SIVcpzPtt strains that contained mosaic pol sequences, pointing to the existence of a divergent SIVcpzPtt lineage that gave rise to SIVgor and HIV-1 group O. Gorillas appear to have acquired this lineage at least 100 to 200 years ago. To examine the biological properties of SIVgor, we synthesized a full-length provirus from fecal consensus sequences. Transfection of the resulting clone (CP2139.287) into 293T cells yielded infectious virus that replicated efficiently in both human and chimpanzee CD4(+) T cells and used CCR5 as the coreceptor for viral entry. Together, these results provide strong evidence that P. t. troglodytes apes were the source of SIVgor. These same apes may also have spawned the group O epidemic; however, the possibility that gorillas served as an intermediary host cannot be excluded.

FIG. 1.

Locations of gorilla and chimpanzee subspecies in west central and east Africa. The natural ranges of western gorillas (Gorilla gorilla, solid green) and eastern gorillas (Gorilla beringei, solid magenta) are shown in relation to the ranges of sympatric central chimpanzees (P. t. troglodytes, hatched green) and eastern chimpanzees (P. t. schweinfurthii, hatched magenta) (16). The western gorilla species is subdivided into the Cross River gorilla (G. g. diehli) on the Cameroonian/Nigerian border and the western lowland gorilla (G. g. gorilla) in southern Cameroon, Gabon, Equatorial Guinea, the Republic of Congo, and the Central African Republic. The eastern species is classified into Grauer's gorilla (G. b. graueri) in the Democratic Republic of the Congo; the mountain gorilla (G. b. beringei) on the border of the Democratic Republic of the Congo, Rwanda, and Uganda; and the Bwindi gorilla (a G. beringei subspecies) in Uganda (the classification of the last is still uncertain). The locations of two study sites (CP and BQ) are indicated. International borders (black lines) and major rivers (blue lines) are also shown.

FIG. 2.

Generation of a replication-competent SIVgor clone. (A) The positions of individual RT-PCR products (open boxes) are shown in relation to the SIVgor provirus. Previously reported BQ664 amplicons are hatched (71). All fragments are drawn to scale, and the length of the assembled population sequence is indicated. Nucleotide sequences are numbered starting at the beginning of the R region in the 5′ LTR (see scale bar). (B) The CP2139 genome was amplified on an independent occasion, using strain-specific primers designed to amplify larger fragments. The resulting second consensus sequence (CP2139.2) differed from the first consensus sequence (CP2139.1) at 54 positions (see Table S3 in the supplemental material). One nucleotide was selected at each of these sites and the resulting master consensus sequence synthesized as three adjoining fragments (schematic on the bottom). Unique restriction enzyme sites allowed directional cloning into a low-copy-number plasmid vector (pBR322-MCS).

FIG. 3.

V3 loop diversity among different ape lentiviruses. The phenetic clustering of SIVgor (red), SIVcpz (black), and HIV-1 (blue) V3 amino acid sequences is shown. Four major V3 clusters are apparent: (i) HIV-1 group M, (ii) the MB/LB strains of SIVcpz, (iii) all other SIVcpz strains (except GAB2) plus HIV-1 group N and SIVgor, and (iv) HIV-1 group O. Dashes indicate gaps introduced for better alignment.

FIG. 4.

Evolutionary relationships of newly derived SIVgor strains. Trees were inferred for four proteomic regions (Gag, Pol1, Pol2, and Env), where Pol1 ends at a recombination breakpoint previously identified in HIV-1 group N, and Pol2 includes downstream Vif sequences. The SIVgor sequences (red) were compared to representatives of SIVcpz (black) and HIV-1 (blue) groups M (U455 and LAI), N (YBF30 and YBF106), and O (ANT70 and MVP5180). The four lowest SIVcpz strains (ANT, TAN1, TAN2, and TAN3) are from eastern chimpanzees; all others are from central chimpanzees. Numbers on internal branches indicate estimated posterior probabilities (only values above 95% are shown). The scale bars represent 0.05 (or 0.1 for Env) amino acid replacements per site.

FIG. 5.

Evolutionary relationships of newly derived SIVgor strains in partial genome regions. Partial Gag, Pol, Vpr-Vpu, and Env sequences corresponding to BQ664 were concatenated. SIVgor sequences are highlighted in red; HIV-1, SIVcpzPtt, and SIVcpzPts strains are as in Fig. 4. Numbers on internal branches indicate estimated posterior probabilities (only values above 90% are shown). The scale bars represent 0.1 replacements per site.

FIG. 6.

Evidence for an SIVcpzPtt lineage ancestral to SIVgor and HIV-1 group O. Trees were inferred for two parts of the Pol1 region in Fig. 4, separated at a putative recombination breakpoint at position 300. Strains are color coded as in Fig. 4. Numbers on internal branches indicate estimated posterior probabilities (only values above 95% are shown). The scale bars represent 0.05 replacements per site. Green, blue, and red circles indicate nodes that were used to calculate the distances shown in Table 1.

FIG. 7.

Evolutionary relationships of SIVgor and SIVcpz strains infecting gorillas and chimpanzees at the CP field site. A tree was inferred for a diagnostic Pol fragment (220 bp). Two SIVcpzPtt strains from the CP field site are boxed. The other strains are color coded as in Fig. 4. Numbers on internal branches indicate estimated posterior probabilities (only values above 95% are shown). The scale bars represent 0.1 replacements per site.

FIG. 8.

Coreceptor usage of SIVgor. JC53-BL cells were pretreated with AMD3100 (inhibitor of CXCR4), TAK779 (inhibitor of CCR5), or both prior to addition of the virus preparations indicated. Virus infectivity is plotted on the vertical axis as a percentage of the untreated control. Virus derived from the replication-competent molecular clones NL4.3 (X4-tropic), YU2C (R5-tropic), and WEAU1.6 (dual tropic) as well as the primary isolates 97US08692A (group O) and YBF30 (group N) were included for control.

FIG. 9.

Replication potential of SIVgor in human and chimpanzee CD4⁺ T cells. The replication kinetics of CP2139.287-derived virus in human (A) and chimpanzee (B) CD4⁺ T cells are shown in relation to those of HIV-1 (SG3, YU2), SIVcpzPtt (MT145, MB897, EK505, and GAB2), and SIVcpzPts (TAN1, TAN2, and TAN3) reference strains (x axis, number of days postinfection; y axis, nanograms of reverse transcriptase [RT] activity per ml of culture supernatant). Similar replication kinetics were observed for CP2139.287-derived virus in CD4⁺ T cells from three additional human and two additional chimpanzee donors.

FIG. 10.

Possible scenarios for the origin of HIV-1 group O. (A) Chimpanzees harboring SIVgor-like viruses infected gorillas and humans independently. (B) Chimpanzees harboring SIVgor-like viruses infected gorillas, which then passed the virus on to humans. (C) Chimpanzees harboring SIVgor-like viruses infected humans, who then passed the virus to gorillas. Support for each scenario would come from the discovery of one of the hypothetical viruses shown in green. In all three trees, nodes marked by circles indicate ancestral viruses that are assumed to have infected chimpanzees.