Characterization of a novel simian immunodeficiency virus with a vpu gene from greater spot-nosed monkeys (Cercopithecus nictitans) provides new insights into simian/human immunodeficiency virus phylogeny

Abstract

In the present study, we describe a new simian immunodeficiency virus (SIV), designated SIVgsn, naturally infecting greater spot-nosed monkeys (Cercopithecus nictitans) in Cameroon. Together with SIVsyk, SIVgsn represents the second virus isolated from a monkey belonging to the Cercopithecus mitis group of the Cercopithecus genus. Full-length genome sequence analysis of two SIVgsn strains, SIVgsn-99CM71 and SIVgsn-99CM166, revealed that despite the close phylogenetic relationship of their hosts, SIVgsn was highly divergent from SIVsyk. First of all, they differ in their genomic organization. SIVgsn codes for a vpu homologue, so far a unique feature of the members of the SIVcpz/human immunodeficiency virus type 1 (HIV-1) lineage, and detailed phylogenetic analyses of various regions of the viral genome indicated that SIVgsn might be a mosaic of sequences with different evolutionary histories. SIVgsn was related to SIVsyk in Gag and part of Pol and related to SIVcpz in Env, and the middle part of the genome did not cluster significantly with any of the known SIV lineages. When comparing the two SIVgsn Env sequences with that of SIVcpz, a remarkable conservation was seen in the V3 loop, indicating a possible common origin for the envelopes of these two viruses. The habitats of the two subspecies of chimpanzees infected by SIVcpz overlap the geographic ranges of greater spot-nosed monkeys and other monkey species, allowing cross-species transmission and recombination between coinfecting viruses. The complex genomic structure of SIVgsn, the presence of a vpu gene, and its relatedness to SIVcpz in the envelope suggest a link between SIVgsn and SIVcpz and provide new insights about the origin of SIVcpz in chimpanzees.

FIG. 1.

Antibody profiles observed in C. nictitans with a line immunoassay (INNO-LIA HIV confirmation; Innogenetics, Ghent, Belgium). The five HIV-1 antigens include synthetic peptides for the exterior envelope glycoprotein (sgp120) as well as recombinant proteins for the transmembrane envelope glycoprotein (gp41), integrase (p31), core (p24), and matrix (p17) proteins. The HIV-2 antigens include synthetic peptides for the exterior envelope glycoprotein (sgp105) as well as recombinant gp36 protein. Plasma samples which recognized at least one HIV antigen with an intensity equal to or greater than the assay cutoff line were scored as positive; samples which exhibited weaker but still visible reactivities with at least two HIV antigens were scored as indeterminant; and samples which yielded no reactivity or only a single band of less than ± intensity were scored as negative. Note that the 3+, 1+, and ± bands evident on the top portions of all test strips control for sample addition (presence of plasma immunoglobulin) and test performance (binding of secondary antibody).

FIG. 2.

(A) Genomic organization of SIVgsn from greater spot-nosed monkeys (C. nictitans). (B) Hydrophobicity profiles of the SIVgsn-99CM71 (a), SIVgsn-99CM166 (b), and SIVcpzANT (c) Vpu proteins were calculated according to Kyte and Doolittle (24). Positive scores are hydrophobic, and the amino acid number is indicated on the x axis. (C) RNA secondary-structure predictions of TAR for SIVgsn-99CM71, SIVgsn-99CM166, SIVsyk, and SIVcpz as generated by the RNA MFOLD 3.0 program by M. Zuker and D. Turner at a 37°C folding temperature. The free energy is expressed in kilocalories per mole.

FIG. 3.

Diversity plot comparing SIVgsn-99CM166 with SIVgsn-99CM71 and representatives of the six major lineages of the primate lentiviruses, i.e., SIVcpz, SIVmnd, SIVsyk, SIVsm, SIVagm, and SIVcol. Protein sequence difference is plotted for windows of 200 amino acids moved in steps of 20.

FIG. 4.

Bootscanning of SIVgsn-99CM166 against the six major SIV lineages. Bootscan analysis was performed on the concatenated nucleotide alignment with the nonoverlapping regions of gag, pol, env, vif, and nef with strains SIVsyk, SIVsun, SIVlhoest, SIVcpzUS, SIVcpzGAB, SIVcpzANT, SIVagmVER155, SIVagmTAN1, SIVmnd1, SIVsmH4, and SIVcol and by grouping them by lineage. Since saturation always occurred at the third codon position for all genes (see Table 2), the bootscan analysis was performed on first plus second codon positions with a sliding window of 500 nucleotides and a 20-nucleotide step increment and 1,000 bootstrap replicates. Below the graph, the bar at the top locates the gene regions, and the colored bar indicates the position of the putative recombination breakpoints in the SIVgsn genome.

FIG. 5.

Unrooted neighbor-joining trees of fragments I, II, III, and IX (see Table 2 and Fig. 4), including 23 SIV/HIV strains of the six major known SIV lineages and the new SIVgsn-99CM166 and SIVgsn-99CM71 isolates (boxed in black). Horizontal branch lengths are drawn to scale, with the bar indicating 0.1 nucleotide replacement per site. Numbers along the branches represent percentage of bootstrap replicates (out of 1,000) for which the sequences at one end of the branch are monophyletic in the neighbor-joining tree. The maximum-likelihood method gives a similar tree topology: an asterisk along a branch indicates that the branch has a P value of <0.001 in the maximum-likelihood analysis. The trees were inferred with first plus second codon position with the TN plus Inv plus G, HKY plus Inv plus G, HKY plus G, or GTR plus G model (see Table 4).

FIG. 6.

Alignment of gp120 of SIVgsn-99CM71, SIVgsn-99CM166, SIVcpzANT, SIVcpzGAB, HIV1-YBF30, HIV1-ANT70, and HIV1-U455. The amino acid sequence of SIVgsn-99CM71 is shown on the top, with variable regions analogous to those observed in previously described SIVs indicated. The CD4 binding domain is shown. An asterisk represents a conserved cysteine residue. Dots indicate amino acid identity at a residue, and dashes indicate gaps introduced to optimize alignment.