Generation of hybrid human immunodeficiency virus utilizing the cotransfection method and analysis of cellular...

Human immunodeficiency viruses (HIV) isolated from infected individuals show tremendous genetic and biologic diversity. To delineate the genetic determinants underlying specific biologic characteristics, such as rate of replication, cytopathic effects, and ability to infect macrophages and T4 lymphoid cells, generation of hybrid HIV using viruses which exhibit distinct biologic features is essential. To develop methods for generating hybrid HIV, we constructed truncated HIV proviral DNA plasmids. Upon digestion with restriction enzymes, these plasmid DNAs were cotransfected into human rhabdomyosarcoma cells to generate hybrid HIV. The hybrid HIVs derived by this method were infectious upon transmission to both phytohemagglutinin-stimulated peripheral blood lymphocytes and established human leukemic T-cell lines. The virus derived from molecular clone pHXB2 (HIVHTLV-III) productively infected CEMx174 cells. On the other hand, molecular clone pARV (HIVSF2)-derived virus did not show productive infection of CEMx174 cells when used as a cell-free virus. The hybrid HIV containing the 3' end of the genome from pARV and the 5' end of the genome from pHXB2 was effective in infecting CEMx174 cells, but the converse hybrid containing 5' pARV and 3' pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef play a role in the ability of the virus to infect a certain cell type. The intracellular ligation method should be useful in the analysis of related and unrelated HIV-1 isolates with common restriction enzyme cleavage sites.