Genomic Stability of Murine Leukemia Viruses Containing Insertions at the Env-3′ Untranslated Region Boundary

Abstract

Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells. We have constructed replication-competent murine leukemia virus (MLV) vectors containing internal ribosome entry site (IRES)-transgene cassettes at the env-3 untranslated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication. A virus containing an insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cycle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated with kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants were detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be maintained at a relative equilibrium. Sequence analysis of these variants identified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert deletion through recombination with an endogenous retrovirus was also observed. When specific sequences involved in these recombination events were eliminated, deletion variants still arose with the same kinetics upon virus passage and by apparently similar mechanisms, although at different locations in the vectors. Our results suggest that while lengthened, insertcontaining genomes can be maintained over multiple replication cycles, preferential deletions resulting in loss of the inserted sequences confer a strong selective advantage.

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

“…34 Deletions in RCR vectors had been observed previously to occur between direct and inverted repeats during reverse transcription. 24 However, this mode of deletion is unlikely here as no repeats were detectable around the deletion site. It is rather likely that shRNA transcription from the U6 promoter interfered with transcription of viral genomes from the 5¢-long terminal repeat.…”

“…22,23 The shRNA expression cassette was inserted between the env gene and the 3¢-long terminal repeat of the MLV genome encoded by pAZE-GFP, 24 resulting in pMLV-shRNA (Figure 1a). We used either the env gene of the 4070A strain to provide a broad tropism for human and murine cells (aMLV-shRNA) or that of the ecotropic strain Moloney (MoMLV-shRNA).…”

“…For the generation of aMLV-shRNA-encoding plasmids PCR fragments were subcloned, sequenceverified and ligated via BsiWI-NotI into pAZE-GFP, 24 thus replacing the IRES-GFP coding sequence of pAZE-GFP with the shRNA expression cassette. The Moloney-derived MoMLV-shRNA viruses were based on plasmid ZAPd-GFP, 24 which does not contain a BsiWI restriction site for cloning of the shRNA expression via BsiWI-NotI.…”

Replicating retroviral vectors mediating continuous production and secretion of therapeutic gene products from cancer cells

“…RCR vectors with notable genomic stablity have been developed recently from amphotropic MLV. [31][32][33] According to these studies, the stability of these RCR vectors is determined by both the proper insertion site and the size of the inserted sequences. Logg et al 31 initially demonstrated that positioning a 1.3-kb cDNA precisely at the boundary between env and the 3 0 UTR regions confers upon an RCR vector the ability to tolerate exogenous sequence.…”

“…It has further been shown that an RCR vector encompassing a a large insertion (1.55 kb) at the same insertion site displayed greatly attenuated replication kinetics and lost inserts even in a single infection cycle, as compared with vectors containing inserts of 1.15 to 1.3 kb or less. 32 Hence, it seems plausible that the replicative fitness of engineered viruses may contribute to a large degree to the greater genomic stability. The results presented here extend these observations by demonstrating that RCR vectors created by strategical selection of a furin site from randomized libraries can be stable over Z12 cycles of infection.…”