Cellular restriction of retroviral particle mediated mRNA transfer

Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; Pediatric Hematology and Oncology, Children's Hospital, Hannover Medical School, Germany; Department of Infection UCL, London, UK; Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

^* To whom correspondence should be addressed. Email: baum.christopher{at}mh-hannover.de.

	Abstract

Analyzing cellular restriction mechanisms provides insight into viral replication strategies, identifies targets for antiviral drug design, and is crucial for the development of novel tools for experimental or therapeutic delivery of genetic information. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription mediate a transient expression of any sequence which replaces the gag-pol transcription unit, a process we call retroviral particle-mediated mRNA transfer (RMT). Here, we further examined the mechanism of RMT by testing its sensitivity to cellular restriction factors and shRNAs. We found that both human TRIM5, and to a lesser extent Fv1, effectively restrict RMT if the RNA is delivered by a restriction-sensitive capsid. While TRIM5 restriction of RMT led to reduced levels of retroviral mRNA in target cells, restriction by Fv1 did not. Treatment with the proteasome inhibitor MG132 partially relieved TRIM5-mediated restriction of RMT but not TRIM5-mediated restriction of wild-type integrating vectors. Finally, cells expressing shRNAs, specifically targeting the retroviral mRNA, inhibited RMT but not reverse transcribing particles. Retroviral mRNA may thus serve as a translation template if not used as a template for reverse transcription. Our data imply that retroviral nucleic acids become accessible to host factors including ribosomes as a result of particle remodeling during cytoplasmic trafficking.