MOLECULAR AND BIOLOGICAL ANALYSES OF QUASI-SPECIES DURING EVOLUTION OF A VIRULENT SIMIAN IMMUNODEFICIENCY VIRUS, SIVSMMPBJ14

A prototypic simian immunodeficiency virus (SIVsmm9), isolated from a naturally infected sooty mangabey (Cercocebus atys), was passaged in vivo in a pig-tailed macaque (Macaca nemestrina) having the identifier PBj. When PBj died of a typical AIDS-like syndrome 14 months after infection, the virus isolated from its tissues was subsequently shown to differ from SIVsmm9 genetically and biologically. Most notably, this isolate, SIVsmmPBj14 (SIV-PBj14), is the most virulent primate lentivirus known: it induces acute disease and death within 6 to 10 days after intravenous inoculation into pig-tailed macaques. Between the time of infection with SIVsmm9 and isolation of SIV-PBj14, isolates were obtained periodically from peripheral blood mononuclear cells of PBj. To establish the temporal relationship between evolution of new biologic properties and fixation of specific mutations in the virus population, these sequential SIV-PBj isolates were characterized for unique properties of SIV-PBj14 that appeared to correlate with acute lethal disease. These properties included the ability to replicate in quiescent macaque peripheral blood mononuclear cells, to activate and induce proliferation of CD4(+) and CD8(+) cells, and to exhibit cytopathicity for mangabey CD4(+) lymphocytes. Consistent with earlier studies, a major change in biologic properties occurred between 6 (SIV-PBj6) and 10 (SIV-PBj10) months, with the SIV-PBj8 quasispecies exhibiting properties of bath earlier and later isolates. Multiple biologic clones derived from the 6-, 8-, and 10-month isolates also exhibited diverse phenotypes. For example, one SIV-PBj10 biologic clone resembled SIVsmm9 phenotypically, whereas three other biologic clones resembled SIV-PBj14. To evaluate genetic changes, proviral DNA of the biologic clones generated from SIV-PBj6, -PBj8, and -PBj10 was amplified by PCR in the U3 enhancer portion of the long terminal repeats (LTR) and the V1 region of env, where the greatest nucleotide diversity between SIVsmm9 and SIV-PBj14 resided. Nucleotide sequence data indicated that all biologically cloned viruses are distinct and that insertions/duplications of 3 to 27 nucleotides (in multiples of three) had accumulated stepwise in the env V1 region, beginning with SIV-PBj8. In addition, one of four SIV-PBj8 biologic clones had a 22-bp duplication in the LTR which is characteristic of SIV-PBj14. When virus mixtures containing different proportions of two SIV-PBj10 biologic clones with opposite phenotypes were tested, the SIV-PBj14 phenotype was clearly dominant, since mixtures with as few as 10% of the viruses being SIV-PBj14-like exhibited all the properties of the lethal isolate. The results suggest that neither the duplication of the NF-KB binding site in the LTR nor the duplications/insertions in env V1 (nor a combination of both mutations) were sufficient to confer the SIV-PBj14 biologic phenotype. However, because some of the unique SIV-PBj14 properties segregate, further analysis of biologically and molecularly cloned viruses derived from these sequential isolates should lead to the identification of viral determinants for specific traits.