Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359))

Abstract

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

FIG. 1

(A to C) Production of SIV p27 antigen in stimulated (A) and resting (nonstimulated) (B) rhesus lymphocyte cultures and in MDM cultures (C). Cultures were infected with equivalent amounts of either SIV_F359 (□), SIV_mac239/YEnef (●), SIV_mac239 (▵), or SIV_mac316 (▴). (D) Production of p27 in culture supernatants of lymphocytes following cocultivation with MDM. Virus production by the parental SIV₈₉₈₀ (⧫) was compared to that by the end-stage variant SIV_F359 (□).

FIG. 2

Photomicrographs of MDM cultures infected with SIV_mac316 (A) and SIV_F359 (B); infection is demonstrated by CD68⁺-p27⁺ double staining. Magnification, ×660.

FIG. 3

Virus load (A and B) and changes in CD4⁺ lymphocyte populations (C and D) of rhesus monkeys WT (A and C) and L52 (B and D) after infection with molecularly cloned SIV_F359. A progressive decline of CD4⁺ T lymphocytes in infected rhesus monkeys (WT and L52) (▪) compared to control monkeys 8637 (□) and 8711 (▵) is seen.

FIG. 4

Abilities of HIV-1_Ba-L, SIV_F359, and SIV₈₉₈₀ to grow on normal human PBMC (wt HuPBMC) versus PBMC homozygous for the Δ32 deletion in the CCR5 receptor gene (Δ32 HuPBMC). The CCR5-restricted HIV_Ba-L isolate could replicate only in wild-type human PBMC, while SIV_F359 and SIV₈₉₈₀ were found to replicate in both Δ32 PBMC and normal human PBMC.

FIG. 5

Comparison of V1 sequences of envelope clones that emerged during the in vivo passages (P1 to P4) to obtain SIV₈₉₈₀, from which the molecular clone SIV_F359 was derived, and V1 sequences of the viral variants (CL1 to CL12) which were present in the original SIV_B670 inoculum (62, 64).

FIG. 6

Amino acid sequence alignments of the V1 envelope region of the predominate clone 12 (CL12) in the prepassage SIV_B670 inoculum. This sequence persisted through the passage as the diversity of the inoculum declined (Fig. 4). The CL12 V1 sequences are compared with the sequences of clones derived from the four different passages (P1 to P4). Clone P4.3 represents the same V1 sequence found in the molecular clone, SIV_F359, and which was originally found in CL12 within the quasispecies found in the prepassage SIV_B670 inoculum.

FIG. 7

(A) Phylogenetic tree based on the SIV_F359 sequence compared with envelope sequences from the parental SIV_B670 and compared to less related SIV_mac251, SIV_sm, and SIV_stm sequences. The tree was constructed using a GTR maximum-likelihood analysis. (B) Phylogenetic analysis based on full-length sequences, comparing the SIV_F359 molecular clone to other SIV, HIV-1, and HIV-2 clones. Phylogenetic trees were constructed by the neighbor-joining method using the PHYLIP program (version 3.5c).

FIG. 8

Comparison of survival of groups of animals infected with the original prepassage SIV_B670 isolate, the postpassage SIV₈₉₈₀ isolate, and the subsequently derived SIV_F359 molecular clone as illustrated by a Kaplan-Meier plot.