After 3-5 years, SIVdelta3, a live attenuated mutant of the simian immunodeficiency virus deleted in vpr, nef, and in the negative regulatory element (NRE) of the viral long terminal repeat (LTR), was 100% pathogenic in neonates. Initially, it remained attenuated in adults, but with time, ca.25% of them became viremic again; one has progressed to AIDS. Molecular analysis revealed that SIVdelta3 lost additional sequences in the 3'LTR; the emergence of shorter viruses was linked to the rate of disease progression in all animals studied. Mutations in env that generate 2 potential new glycosylation sites also arose independently in different animals with AIDS. By direct cloning from tissue of an SIVdelta3-infected, diseased infant, we isolated a full-length infectious, pathogenic proviral clone, termed SIVdelta3+.
The Specific Aims are to: 1. Assess functional alterations in SIVdelta3+ in comparison to parental SIVdelta3. We will focus first on the effects of congruous mutations in the LTR and in env. Did the shortened viral LTR lose negative control elements? Are the env gene mutations associated with a change in viral tropism? Is SIVdelta3+ CD4-independent? Is co-receptor usage altered? 2. Follow existing cohorts of SIVdelta3-infected adults and infants prospectively for the emergence of the same and/or other congruous mutations. Are such mutations linked to disease progression? What are the biological characteristics of uncloned virus collected from animals at various time points during disease progression? 3. Test the stability of the congruous mutations in adult and infant macaques inoculated with molecularly cloned SIVdelta3+ proviral DNA. 4. Introduce the congruous mutations identified in the previous Specific Aims by site-directed mutagenesis into the parental SIVdelta3 genome and test the resulting virus in vitro and in vivo. Will the congruous mutations be sufficient to confer increased virulence to the viral recombinants? The proposed work is significant because it seeks to dissect viral factors associated with increased virulence, using molecularly cloned pro-viruses with striking biological differences that evolved in vivo.
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