The possible contribution of HIV-1-induced syncytia to the generation of intersubtype recombinants in vitro

To develop a method for single syncytia isolation and delineate the possible contribution of syncytia to intersubtype recombination. We dually infected whole peripheral mononuclear blood cells with subtype A and D viruses and studied syncytia in vitro and developed a method to isolate individual syncytia to further study HIV variants/dual infections, viral isolation, proviral copies in single syncytia and possible intersubtype recombination in dual cultures containing syncytia using real time PCR. Cell culture-based single syncytia isolation, PCR and cloning to determine the nature of HIV variants and real-time PCR to determine proviral copies per individual syncytium and intersubtype recombination in dual cultures. Viral coculture from single syncytia and p24 antigen determination for assessing viral replication in vitro. Our results show the feasibility that not only can single syncytia be successfully isolated, but the viruses from individual syncytia can also be grown in vitro. They also demonstrate the ability of single syncytia to bring diverse HIV-1 subtypes together along with the possible contribution to intersubtype recombination in vitro. Up to 40% of single syncytia harbored both input HIV-1 subtypes and single syncytium could harbor as many as 2000 proviral DNA copies, which exceeds the limit seen in a single cell. These analyses are unique in experimentally confirming the previously held belief that single syncytia can harbor multiple HIV strains and that they can serve as a breeding ground for heterozygous virions and this may contribute toward viral diversity and intersubtype recombination.