Adaptive Evolution of a Tagged Chimeric Gammaretrovirus: Identification of Novel cis-Acting Elements that Modulate Splicing

Abstract

SummaryRetroviruses are well known for their ability to incorporate envelope proteins from other retroviral strains and genera and even from other virus families. This characteristic has been widely exploited for the generation of replication-defective retroviral vectors, including those derived from murine leukemia virus (MLV), bearing heterologous envelope proteins. We desired to investigate the possibility of "genetically" pseudotyping replication-competent MLV by replacing the native env gene in a full-length viral genome with that of another gammaretrovirus. We previously developed replication-competent versions of MLV that stably transmit and express transgenes inserted in the 3′ untranslated region of the viral genome. In one such tagged MLV expressing green fluorescent protein, we replaced the native env sequence with that of gibbon ape leukemia virus (GALV). Although the GALV Env protein is commonly used to make high titer pseudotypes of MLV vectors, we found that the env replacement greatly attenuated viral replication. However, passage of cells exposed to the chimeric virus resulted in selection of mutants exhibiting rapid replication kinetics and different variants arose in different infections. Two of these variants had acquired mutations at or adjacent to the splice acceptor site and three others had acquired dual mutations within the long terminal repeat. Analysis of the levels of unspliced and spliced viral RNA produced by the parental and adapted viruses showed that the mutations gained by each of these variants functioned to reverse an imbalance in splicing caused by the env gene substitution. Our results reveal the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and demonstrate that tagging of the retroviral genome with an easily assayed transgene can be combined with in vitro evolution to efficiently generate and screen for replicating mutants of replicationimpaired recombinant viruses.

Splicing mediates the activity of four putative cellular internal ribosome entry sites

“…MLV must maintain a balance between its full-length genomic transcript and spliced env transcript to undergo productive replication. We previously found that MLV is highly sensitive to modifications to its genome that perturb normal splicing of the viral transcript and that attenuation of replication can serve as a useful indicator of aberrant splicing (23). In stark contrast to the results obtained with the 5Ј ss-depleted dicistronic constructs, the proviruses containing the eIF4G, NRF, Rbm3, and XIAP sequences produced exceedingly high levels of GFP upon transfection (Fig.…”

“…As an approach to producing such mutations, we serially passaged the viruses containing these IRESs to evolve mutants in which the splice sites had been inactivated through natural selection. We previously used this method to isolate rapidly replicating mutants of a retrovirus that had been crippled by an oversplicing defect (23). From five passaged isolates of the NRF and Rbm3 viruses, we identified three unique mutants, each of which possessed a single or double point mutation at or adjacent to the IRES 3Ј ss (SI Fig.…”

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

“…The pAZE-GFP plasmid is a biologically active clone obtained from the laboratory of Christopher Logg [50]. It contains LTRs and gagpol derived from Moloney murine leukemia virus and an envelope gene derived from amphotropic MLV.…”

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

“…The alloCTL were generated by one-way MLTR according to methods previously published 9 . Effector alloCTL were harvested 4-5 days after one-way MLTR and transduced with RRV-GS4-EMD (Figures 3 and 5) or RRV-ACE-EMD (Figure 6) 10,11 . Higher transduction levels were achieved with the GS4 vector utilizing a Gibbon ape leukemia virus envelope than with the amphotropic ACE vector.…”

“…We also used transduction with viral vectors encoding for fluorescent proteins as an additional way to visualize the cells. For certain assays, we transduced the alloCTL with retroviral replicating vectors (RRV) coding for Emerald Green (EMD) fluorescent protein 10,11 ; for others, tumor cells were transduced with lentiviral vectors coding for mStrawberry.…”