Characterization of novel simian immunodeficiency viruses from red-capped mangabeys from Nigeria (SIVrcmNG409 and -NG411)

Abstract

Two novel simian immunodeficiency virus (SIV) strains from wild-caught red-capped mangabeys (Cercocebus torquatus torquatus) from Nigeria were characterized. Sequence analysis of the fully sequenced SIV strain rcmNG411 (SIVrcmNG411) and gag and pol sequence of SIVrcmNG409 revealed that they were genetically most closely related to the recently characterized SIVrcm from Gabon (SIVrcmGB1). Thus, red-capped mangabeys from distant geographic locations harbor a common lineage of SIV. SIVrcmNG411 carried a vpx gene in addition to vpr, suggesting a common evolutionary ancestor with SIVsm (from sooty mangabeys). However, SIVrcm was only marginally closer to SIVsm in that region than to any of the other lentiviruses. SIVrcm showed the highest similarity in pol with SIVdrl, isolated from a drill, a primate that is phylogenetically distinct from mangabey monkeys, and clustered with other primate lentiviruses (primarily SIVcpz [from chimpanzees] and SIVagmSab [from African green monkeys]) discordantly in different regions of the genome, suggesting a history of recombination. Despite the genetic relationship to SIVcpz in the pol gene, SIVrcmNG411 did not replicate in chimpanzee peripheral blood mononuclear cells (PBMC), although two other viruses unrelated to SIVcpz, SIVmndGB1 (from mandrills) and SIVlhoest (from L'Hoest monkeys), were able to grow in chimpanzee PBMC. The CCR5 24-bp deletion previously described in red-capped mangabeys from Gabon was also observed in Nigerian red-capped mangabeys, and SIVrcmNG411, like SIVrcmGB1, used CCR2B and STRL33 as coreceptors for virus entry. SIVrcm, SIVsm, SIVmndGB1, and all four SIVlhoest isolates but not SIVsun (from sun-tailed monkeys) replicated efficiently in human PBMC, suggesting that the ability to infect the human host can vary within one lineage.

FIG. 1

Serologic identification of SIV infection in wild-caught red-capped mangabeys by radioimmunoprecipitation of SIVsm proteins. Lanes contain SIV antigens immunoprecipitated by plasma samples from 13 red-capped mangabeys and are identified individually by animal numbers; RCM409 and RCM411 plasma samples show a positive reaction with SIVsm envelope proteins. The last two lanes on the right show plasma from an uninfected monkey and plasma from an SIVsm-infected pigtailed macaque. The positions of molecular weight markers (in thousands) are shown to the left, and SIVsm proteins are identified to the right. CEMx174 cells infected with SIVsmE660 were labeled overnight with l-[³⁵S]methionine and l-[³⁵S]cysteine (Amersham), lysed, and precipitated with plasma from wild-caught red-capped mangabeys from Nigeria.

FIG. 2

Genomic organization of SIVrcm compared to other representative primate lentiviruses. The genome organizations are shown schematically for SIVrcm, SIVsm, HIV-2, SIVmac, and SIVstm (top panel), SIVcpz and HIV-1 (middle panel), and SIVagm, SIVsyk, SIVlhoest, SIVsun, SIVmnd, and SIVcol (bottom panel).

FIG. 3

RNA secondary structures predictions of TAR. Secondary structure predictions of SIVrcm and other primate lentiviruses as generated by the RNA MFOLD 3.0 program by M. Zuker and D. Turner at a 37°C folding temperature (http://w.w.w.mfold2.wustl.edu/∼mfold/rna/form1.cgi [29, 47]). The free energy is expressed in kilocalories per mole. The different viral strains are as indicated (for GenBank accession numbers of virus strains, see Material and Methods).

FIG. 4

Similarity plots comparing parts of gag and pol of SIVrcmNG411 with those of SIVrcmNG409, SIVdrl, and representatives of the six major lineages of primate lentiviruses, SIVcpz, SIVsmm, SIVagmSab, SIVagmVer, SIVlhoest, SIVsyk, and SIVcol.

FIG. 5

Phylogenetic trees and similarity plot analysis of primate lentiviral full-length genomes. (A) The primate lentiviral genomes were aligned as described in Materials and Methods, columns containing gaps were removed from the alignment, and the resulting gap-stripped alignment was subdivided into seven regions based on the similarity relationship of the SIVrcm sequence to other sequences as detected by SimPlot (Fig. 6B). Each region was then used to build a phylogenetic tree as described in Materials and Methods. The sequences used are all available from http://w.w.w.hiv-web.lanl.gov using either the common names or accession numbers (see Materials and Methods). Leaves on each tree are colored similarly to the shades of colors used in the SimPlot. Although many nodes in the trees have 100% bootstrap support, only three nodes (in trees 3, 5, and 6) supported the hypothesis of a recombination event or events involving the lineage leading to the RCM.NG411 sequence, and they are indicated by arrows. In the other four trees, the bootstrap support for RCM.NG411 belonging to a clade including other lineages was less than 50%. (B) The gap-stripped sequence alignment described for panel A was analyzed with the SimPlot program written by Stuart Ray using a window size of 500 bases and a step increment of 20 bases. The nucleotide similarity score computed by SimPlot is a corrected phylogenetic distance/similarity score within the window and corrects for multiple mutations per site via the Kimura two-parameter model, which counts transitions differently from transversions (Ts/Tv ratio set to 1.7 in this figure). Thus, the similarity scores produced by SimPlot can be compared to the phylogenetic distances in panel A, although different models of evolution were used (Kimura 2-parameter in B: F84 maximum likelihood with site-specific rates in panel A).

FIG. 6

Replication of SIVrcm in human and chimpanzee PBMC. (A) Infection of PHA-stimulated human PBMC from two different donors with a lack of the 32-bp deletion in CCR5. A total of 2.5 × 10⁶ PHA-stimulated human PBMC were infected in 12-well plates with a virus amount corresponding to 500,000 cpm of RT activity. (B) Infection of PHA-stimulated chimpanzee PBMC from two different donors. A total of 1 × 10⁷ cells were infected in T25 flasks with a virus amount corresponding to 100,000 cpm of RT activity. Virus stocks used are as indicated.

FIG. 7

Evaluation of coreceptor usage of SIVrcm in the GHOST cell assay. Human osteosarcoma cells, transfected with HIV-2 LTR-GFP, CD4, and different HIV coreceptors were infected with SIV or HIV in the presence of polybrene as indicated. (A) GFP expression following transactivation by tat was measured on days 2, 4, 6, and 9 after infection. The green fluorescence is quantified as percent GFP-positive cells. The parental GHOST cell line, expressing CD4 only, served as a negative control. (B) The course of infection of GHOST cell lines as monitored by RT activity in culture supernatants.

FIG. 7

Evaluation of coreceptor usage of SIVrcm in the GHOST cell assay. Human osteosarcoma cells, transfected with HIV-2 LTR-GFP, CD4, and different HIV coreceptors were infected with SIV or HIV in the presence of polybrene as indicated. (A) GFP expression following transactivation by tat was measured on days 2, 4, 6, and 9 after infection. The green fluorescence is quantified as percent GFP-positive cells. The parental GHOST cell line, expressing CD4 only, served as a negative control. (B) The course of infection of GHOST cell lines as monitored by RT activity in culture supernatants.

FIG. 8

Analysis of CCR5 alleles of RCM. Separation of PCR products generated with primers spanning a 24-bp deletion in red-capped mangabey CCR5 (deletion from base pairs 439 to 462) in a 0.9% agarose gel. A fragment of 213 bp is indicative of the wild-type CCR5 allele, and a 189-bp fragment is indicative of CCR5 with a 24-bp deletion. Amplification of both fragments indicates heterozygosity for the CCR5 deletion. SIV-positive animals are indicated with stars.