Self-limiting infection by int/nef-double mutants of simian immunodeficiency virus

Simian immunodeficiency virus (SIVmac) infectious for rhesus monkeys was altered by site-directed mutagenesis of genes influencing in vivo replication and persistence with the long-term goal to develop attenuated lentiviruses with limited replication capacity in vivo. Double mutants of SIVmac (termed delta-int 1 to 3) were generated by introducing frameshift and deletion mutations into the nef gene and into the pol gene region coding for the integrase protein. Delta-int/delta-nef viruses formed after transfection of CD(4+)-lymphocyte cell lines were unable to establish sustained replication. In contrast, both wild-type SIVmac and mutant SIVmac delta-nef (coding for a truncated NEF protein and a wild-type INT protein) replicated continuously and at a comparable rate. However, a transient and self-limiting infection of the C8166 T-cell line was observed subsequent to transfection of double mutant proviruses into HeLa-tat-III cells. Viruses attenuated by int/nef-double mutation were able to enter the T-cells, initiate synthesis of viral DNA as shown by PCR amplification of closed circular episomes, and express viral antigens in infected cells as demonstrated by immunocytochemical staining. Integration of the int mutant viruses into the chromosome was completely inhibited. Episomal viral DNA was detectable in the infected cells for up to 2 weeks, after which it disappeared. Thus, SIVmac attenuated by int and nef mutation established a transient infection of permissive cells resulting in the expression of viral antigen from episomal viral DNA over a limited period of time.