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To achieve specific gene transfer into human CD4(+) cells, murine leukaemia virus (MLV)-based pseudotype vector particles were generated employing Env variants derived from human or simian immunodeficiency virus (HIV-1 or SIVagm). Here, we describe the generation of full-length onco/lentivirus hybrid genomes comprising components of MLV and HIV-1 or SIVagm, respectively, to assess the possibility of replication-competent hybrid virus formation. The env reading frame of an infectious molecular clone of MLV was replaced with the analogous coding regions of HIV-1 or SIVagm encompassing the env gene and accessory genes. Resulting MLV/HIV-1 or MLV/SIVagm hybrid genomes were transfected into 293T cells. Expression of viral proteins and budding of retroviral particles was shown by specific immunostaining and electron microscopy. The viral particles mediated CD4- and co-receptor-specific infection of human cells as demonstrated by PCR and immunostaining in the respective target cells. However, no productive infection resulting in the generation of infectious virus was detected in these cells. Thus, these onco/lentivirus hybrids, although able to initiate single-round infection, were not replication competent. Thus, MLV-based pseudotype vectors carrying Env variants of HIV-1 or SIVagm are not prone to form replication-competent retroviruses, suggesting a favourable safety profile for MLV-based CD4-specific pseudotype vectors.

Genetic engineering of onco/lentivirus hybrids results in formation of infectious but not of replication-competent viruses