Simian-human immunodeficiency virus (SHIV) containing the nef/long terminal repeat region of the highly virulent SIVsmmPBj14 causes PBj-like activation of cultured resting peripheral blood mononuclear cells, but the chimera showed No increase in virulence

Abstract

SIVsmmPBj14 is a highly pathogenic lentivirus which causes acute diarrhea, rash, massive lymphocyte proliferation predominantly in the gastrointestinal tract, and death within 7 to 14 days. In cell culture, the virus has mitogenic effects on resting macaque T lymphocytes. In contrast, SIVmac239 causes AIDS in rhesus macaques, generally within 2 years after inoculation. In a previous study, replacement of amino acid residues 17 and 18 of the Nef protein of SIVmac239 with the corresponding amino acid residues of the Nef protein of SIVsmmPBj14 yielded a PBj-like virus that caused extensive activation of resting T lymphocytes in cultures and acute PBj-like disease when inoculated into pig-tailed macaques. This study suggested that nef played a major role in both processes. In this study, we replaced the nef/long terminal repeat (LTR) region of a nonpathogenic simian-human immunodeficiency virus (SHIV), SHIVPPc, with the corresponding region from SIVsmmPBj14 and examined the biological properties of the resultant virus. Like SIVsmmPBj14, SHIVPPcPBjnef caused massive stimulation of resting peripheral blood mononuclear cells (PBMC), which then produced virus in the absence of extraneous interleukin 2. However, when inoculated into macaques, the virus failed to replicate productively or cause disease. Thus, while these results confirmed that the nef/LTR region of SIVsmmPBj14 played a major role in the activation of resting PBMC, duplication of the cellular activation process in macaques may require a further interaction between nef and the envelope glycoprotein of simian immunodeficiency virus because SHIV, containing the envelope of human immunodeficiency virus type 1, failed to cause activation in vivo.

FIG. 1

Schematic diagram of the construction of the 3′ halves of the SHIV_PPc and SHIV_PPcPBjnef genomes. The origin of the DNA from macaque PPc that was used to amplify specific regions of the viral genome and the restriction endonuclease sites used to construct chimeric viruses SHIV_PPc and SHIV_PPcPBjnef are shown. LN, lymph node.

FIG. 2

Immunoprecipitation analyses demonstrate that Nef is synthesized in SHIV_PPc-infected cells. SHIV_PPcΔnef, SHIV_PPc, SHIV_PPcPBjnef, or SIV_SmmPBj14 (10³ TCID₅₀ each) was used to inoculate C8166 cells. At 4 days postinoculation, cells were starved for methionine and cysteine and radiolabeled with 200 μCi of [³⁵S]methionine-[³⁵S]cysteine for 18 h. Cell lysates were prepared as previously described (27, 28), and SHIV proteins were immunoprecipitated with a rabbit anti-Nef serum and protein A-Sepharose as described in the text. Immunoprecipitates were washed three times in radioimmunoprecipitation assay buffer, and samples were denatured by boiling in SDS-PAGE sample-reducing buffer. Proteins were separated by SDS-PAGE (8.5% gel) and visualized by standard autoradiographic techniques. Lanes: 1, proteins immunoprecipitated from uninfected C8166 cells; 2, proteins immunoprecipitated from C8166 cells inoculated with SHIV_PPc with the nef gene deleted; 3, proteins immunoprecipitated from C8166 cells inoculated with SHIV_PPc; 4, proteins immunoprecipitated from C8166 cells inoculated with SHIV_PPcPBjnef; 5, proteins immunoprecipitated from C8166 cells inoculated with SIV_smmPBj14.

FIG. 3

Photomicrographs of C8166 cultures following inoculation with various SHIVs. C8166 cultures were inoculated with 10³ TCID₅₀ of SHIV-4, SHIV_PPc, or SHIV_PPcPBjnef. At the peak period of cytopathology (4 days), the cultures were photographed. (A) Uninfected C8166 culture; (B) SHIV-4-inoculated culture; (C) SHIV_PPc-inoculated culture; (D) SHIV_PPcPBjnef-inoculated culture. Magnifications, ×200 (A) and ×100 (B to D).

FIG. 4

Mitogenic activity (A) and replication (B) of SHIV_PPcPBjnef in resting PBMC. (A) Resting PBMCs (10⁷) were inoculated with 10³ TCID₅₀ of SHIV_PPc (▪), SHIV_PPcPBjnef (▵), or SHIV_KU-1 (•), and mitogenic activity was monitored by determination of [³H]thymidine incorporation until day 16. □, control (uninoculated resting PBMC) cultures. (B) Replication of SHIV_PPc PBjnef and SHIV_PPc in cultures of resting PBMC. Resting PBMC (10⁷) were inoculated with SHIV_PPc (□) or SHIV_PPcPBjnef (▵), and virus replication was assessed by standard p27 antigen capture assay.