Cellular Restriction of Retrovirus Particle-Mediated mRNA Transfer

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 retrovirus particle-mediated mRNA transfer (RMT). Here, we further examined the mechanism of RMT by testing its sensitivity to cellular restriction factors and short hairpin RNAs (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. Finally, cells expressing shRNAs specifically targeting the retroviral mRNA inhibited RMT particles, 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.Retroviruses enter cells in a receptor-mediated manner, following which a reverse transcription (RT) complex is formed in the cytoplasm to reverse transcribe the genomic mRNA into double-stranded DNA. During the completion of RT, a hybrid virus-cellular nucleoprotein structure known as the preintegration complex (PIC) is formed. Eventually, active transport of the PIC into the nucleus or dissolution of the nuclear membrane during mitosis allows the viral integrase to integrate the viral double-stranded DNA into chromosomal cellular DNA (35). We have previously shown that retroviral vector mutants that are unable to initiate RT of their capped, plus-stranded mRNA genomes mediate a transient expression of the sequences cloned into the gag-pol-equivalent position of the vector genome (8). Particles conferring this activity required the presence of the retroviral mRNA packaging signal within the vector sequence, as well as the expression of both Gag and Env in the vector packaging cell, but not reverse transcriptase. We refer to this previously unexplored aspect of the retrovirus life cycle as retrovirus particle-mediated mRNA transfer (RMT). Using replication-defective retroviral vectors in which the gene of interest is cloned in the position of the gag reading frame, RMT can be exploited as a novel approach for the transient expression of a gene of interest (8).The analysis of cellular restriction factors that belong to the innate immune response against retroviruses may provide further insights into the mechanisms of RMT. The cellular restriction factor Fv1 (Friend virus susceptibility factor 1) h...

Protein transduction from retroviral Gag precursors

“…As envelope proteins can be modified to deliver cytokine signals (47), it may be possible to engineer cells using multifunctional retroviral particle preparations, which elicit a cascade of immediate, transient, and permanent effects (48). Underlining the versatility of this concept, intermediate steps of the retroviral life cycle can be exploited to deliver episomal DNA (14,15,49) or mRNA (13,50).…”

“…Interestingly, the transgene-dependent expression of GFP (ILV and NILV) started to accumulate beyond 10 h posttransduction, exactly when the polyprotein-mediated transduction began to decrease. Compared with the polyprotein-mediated delivery, NILV-transduced cells showed a much longer duration of expression (declining 50 h posttransduction) with persistence in a subset of cells, as described (13)(14)(15). Thus, rapid onset and full reversion was only obtained with protein transduction.…”

Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges

“…Several controls, including the use of shRNAs directed against the incoming genome, confirmed the viral genome source of RMT. 71,72 RPT. Using retrovirus-mediated Flp transfer, Voelkel et al 76 demonstrated in a pilot study that gammaretroviral particles tolerate the incorporation of a foreign protein at several positions along their Gag-or Gag-Pol precursors.…”

“…75 RMT. MLV-based vectors encoding Cre-recombinase have been used by Galla et al 72 for transient, nontoxic, and highly efficient recombination of mouse and human Cre indicator cell lines. RMT vectors harbor a disabled primer binding site that was designed not to match any naturally occurring tRNA molecule, the primer necessary for initiation of retroviral reverse transcription.…”

“…These processes, which rely on systematic blocks within the regular RV life cycle, are covered by the term retroviral "pseudotransduction." 71,72 The following subsections will first address RET before RMT, and RPT will bridge this topic with a paragraph on the cell-specific delivery of recombinases.…”