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

Abstract

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.

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

“…This infectivity is clearly lower than that usually observed for retroviral particles, where 1 in 100 to 1,000 particles is infective (46). Nevertheless, infectivity is clearly codetermined by the glycoproteins used for pseudotyping retroviral vector particles (48,49) and may vary by at least two logs (49). Accordingly, glycoproteinspecific differences have been observed also in this study when VSV-G or MV-H/F expression plasmids were titrated against the other constituents of PTVs during establishment of PTV generation (Fig.…”

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“…J02255) and an additional Sfi I-site at position 5389 removed by mutation. The start codon of MLV env (position 5777) was deleted to allow translation to start at the inserted GFP sequence [16]. We replaced GFP with RFP, which was introduced as a Sfi I-Cla I fragment.…”

Intracellular assembly and budding of the Murine Leukemia Virus in infected cells

“…It is not clear what determines the incidence of intracellular versus cell surface assembly. Indeed, numerous EM analyses performed with transfected MLV Gag or reconstituted viruses, usually in human (293T), monkey (Cos), or hamster (BHK21) cell lines, described exclusive budding at the plasma membrane [ 26 - 28 ]. At the opposite, only two studies (ours and [ 23 ]) showed the coexistence of intracellular and cell surface in chronically infected cells.…”