FIG. 4.

Integration of viral DNA. P4p56 cells were exposed to WT or IN-mutated (L, Q, LQ, and N) virions in amounts adjusted to ensure equivalent levels of p24. At 24 h following infection, DNA was prepared and analyzed by PCR to visualize integrated and total viral DNA. Integrated viral DNA (top) was amplified by nested PCR. In the first round of PCR, a 5′ primer from the conserved human Alu sequence and a 3′ primer from the conserved HIV-1 LTR sequence were used. This round amplifies both cellular DNA upstream of the integration site and integrated HIV-1 LTR. An aliquot (1/400) of the first PCR product was further subjected to the second round of PCR by using nested HIV-1 LTR-specific primers. To verify that only integrated viral DNA was amplified by the two-round procedure, control reactions (including the 1/400 dilution step) were performed in which the primers and the enzyme were omitted in the first PCR (not shown). Total viral DNA (bottom) was amplified using primers from the HIV-1 LTR sequence. Mock-infected cells were similarly analyzed as a negative control (lane CTRL).