RESUMO
A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.
Assuntos
Vetores Genéticos/genética , Vírus da Leucemia Murina de Moloney/fisiologia , Provírus/fisiologia , Proteínas do Envelope Viral/química , Replicação Viral/genética , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , DNA Viral/análise , Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/genética , Vírus do Sarcoma Murino de Moloney/genética , Vírus do Sarcoma Murino de Moloney/fisiologia , Reação em Cadeia da Polimerase , Recombinação Genética/genética , Recombinação Genética/fisiologia , Segurança , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética , Replicação Viral/fisiologiaRESUMO
Certain subclones (designated as minus clones) of the promonocytic U937 cell line do not support efficient infection and fusion mediated by T cell line adapted (TCLA) X4 HIV-1 gp120-gp41 (Env) although the CXCR4 and CD4 concentrations at their surfaces are similar to those at the surfaces of clones susceptible to HIV-1 entry (plus clones) (H. Moriuchi et al., J. Virol. 71, 9664-9671, 1997). To test the hypothesis that inefficient formation of gp120-CD4-CXCR4 complexes could contribute to the mechanism of resistance to Env-mediated fusion in the minus clones, we incubated plus and minus cells with HIV-1 LAI gp120 and coimmunoprecipitated CD4 by using anti-CXCR4 antibodies. The gp120 induced inefficient coimmunoprecipitation of CD4 in the minus clones but not in the plus ones. Overexpression of CD4 resulted in significant restoration of the minus clones' susceptibility to fusion in parallel with an increase in the amount of the gp120-CD4-CXCR4 complexes. These results not only suggest that the resistance to TCLA X4 HIV-1 entry in the U937 minus clones is due to the inability of these cells to efficiently form complexes among CD4, gp120, and CXCR4, but also provide a direct evidence for the correlation between fusion and the cell surface concentration of the complexes among CXCR4, CD4, and gp120. These data and similar recent observations in macrophages suggest that inefficient complex formation among CXCR4, CD4, and gp120 could be a general mechanism of cell resistance to gp120-gp41-mediated fusion and a major determinant of HIV-1 evolution in vivo.