7th International AIDS Conference Florence, Italy — June 16-21, 1991

Int Conf AIDS 1991 Jun 16-21; 7:28 (abstract no. M.A.26)
LaBranche CC, Bugelski PJ, Hart TK, Hoxie JA; Univ. of Pennsylvania, Philadelphia, PA

OBJECTIVE: To identify the determinants of cytopathogenesis in a cytopathic variant of molecularly-derived SIVmac251.

METHODS: SIVmac251 has been shown to infect SUP-T1 cells without killing, fusion, or CD4 down-modulation. A cytopathic variant of this virus arose spontaneously during long-term culture. The parental and variant viruses were compared by time-course analysis of equivalent inocula onto SUP-T1 cells. Modulation of CD4 from the surface of infected cells was determined by flow cytometry (FACS) using monoclonal antibodies (mAb) to CD4. The presence of gp120 (SU) on the surface of infected cells and the ability of SU to bind CD4 were determined by FACS using polyclonal antiserum to SIVmac and fluoresceinated CD4 (F-CD4), respectively. Viral proteins were analyzed by SDS-AGE and western blot or radioimmunoprecipitation of viral and infected cell lysates.

RESULTS: In contrast to prototypical strains of HIV-1 and HIV-2, SIVmac251 derived from the infectious molecular clone K28 (NC-MAC) infected SUP-T1 cells slowly and without cytopathic effect or down-modulation of CD4. However, the cytopathic variant (CP-MAC) exhibited a cytopathic phenotype in SUP-T1 similar to that of HIV-1 and -2; infection was rapid and down-modulated CD4 from the surface of infected cells. Western blots using a mAb to the transmembrane protein (TM) showed that the TM of CP-MAC migrated at 28 kDa, as opposed to 32 kDa for NC-MAC. This mAb also identified a 77kDa species, likely a stable TM dimer, in NC-MAC but not in CP-MAC. F-CD4 bound to SU expressed on the surface of CP-MAC-infected cells with an affinity similar to that observed for HIV-1; this is contrary to published reports showing a reduced affinity of SIV(MAC) SU for soluble CD4. Electron micrographs revealed that CP-MAC virions possessed a significantly greater number of envelope spikes as opposed to NC-MAC virions.

CONCLUSIONS: The cytopathic CP-MAC expresses a TM protein that is smaller than that of the NC-MAC. While other factors may also be involved, changes in TM may profoundly affect processing and stability of the viral envelope and the cytopathicity of the virus. In addition, because these two viruses were derived from a single infectious molecular clone, sequence comparison will allow us to identify the genetic determinants of cytopathogenesis for P-MAC in SUP-T1 cells and establish their biological effects.

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