HIV DNA integration during cell-to-cell transmission of infection: evidence for partially integrated DNA structures in acutely infected cells

A one-step cell-to-cell transmission model of human immunodeficiency virus (HIV) infection was used to study viral DNA integration in the early phase of viral replication. Co-culturing H3B cells as virus donors with CD4+ Hut7 8 recipient cells in a ratio of 1:4 produced a synchronous, one-step viral infection with de novo synthesis of unintegrated HIV DNA within 4 h p.i., w hich subsequently integrates in the host genomic DNA to form provirus. To s tudy the kinetics of viral DNA integration, cellular chromosomal DNA was is olated at different times after co-culturing and extensive electrophoresis was used to remove residual unintegrated viral DNA. Removal of contaminatin g, unintegrated viral DNA in the purified chromosomal DNA fraction was conf irmed by various experiments. When purified chromosomal DNA (free of contam inating unintegrated viral DNA) - from the mix of acutely infected cells - was digested with restriction enzymes KpnI, BamHI or PstI and analysed by S outhern blot hybridization, integration of viral DNA into chromosomal DNA w as first observed at 8 h p.i. and was essentially complete by 72 h p.i. In addition, evidence was found for a relatively stable, partially integrated HIV DNA structure within the chromosomal DNA, that was first detectable at 8 h p.i. and did not become fully integrated until 72 hours post infection.