Fig. 5.

Lack of effect of human Topo I on the stability of HIV-1 genomic RNA (A) or on viral processing (B), assembly (C), or entry (D). (A) COS-1 cells were cotransfected with pNL-Luc-HXB and either an expression vector for human WT, HuE236D/N237S mutant, or African green monkey WT Topo I proteins or the corresponding empty vector (pcDNA). The amount of HIV-1 RNA remaining at the indicated times after exposure of the transfected cells to actinomycin D is expressed as a percentage of that present at time 0. Data are means ± SD of three independent experiments. (B) Human (293T) or African green monkey (COS-1) cells were cotransfected with pNL-Luc-HXB and either the human Topo I expression vector or empty vector. Cell lysates were subsequently subjected to immunoblot analysis either with sera of patients infected with HIV-1 for detection of gp160 and gp120 (Top and Middle) or with a mixture of monoclonal antibodies to p24 and to p17 for detection of Pr55^gag, p41, p24, and p17 (Bottom). Nontransfected cells were similarly analyzed as a control (-). (C) 293T and COS-1 cells were cotransfected with pNL-Luc-HXB and either the human Topo I expression vector (+) or empty vector (-). The amount of p24 in the culture supernatants was subsequently determined and expressed relative to the value for 293T cells expressing recombinant human Topo I as a measure of virion release efficiency. Data are means of triplicates from a representative experiment. (D) 293T or COS-1 cells were cotransfected with pNL-Luc-E^–R⁺, a VSV-G expression vector, and either the human Topo I expression vector (+) or empty vector (-), after which the infectivity of resulting viruses was determined with MAGIC5 cells. Data are means ± SD of triplicates from a representative experiment. Recombinant human Topo I in the producer cells was also detected by immunoblot analysis with antibodies to the Xpress epitope (shown below the histogram).