Efficient Antisense Targeting of Human Immunodeficiency Virus 1 (HIV-1) requires the Rev Response Element (RRE) and Rev Protein

Ward, Alex Michael, Department of Microbiology, University of Virginia
Hammarskjold, Lou, Department of Microbiology, University of Virginia
Rekosh, David, Department of Microbiology, University of Virginia
Beyer, Ann, Department of Microbiology, University of Virginia
Dutta, Anindya, Department of Biochemistry and Molecular Genetics, University of Virginia
Kedes, Dean, Department of Microbiology, University of Virginia

An HIV-1-based vector expressing antisense RNA to env is currently in clinical trials. This vector has shown a remarkable ability to inhibit HIV-1 replication, in spite of the fact that therapeutic use of antisense RNAs has generally been disappointing. We decided to further analyze the basis for why the antisense inhibition is so efficient. To determine if co-targeting to common RNA export pathways made a significant contribution to efficient antisense inhibition, we constructed plasmid-based HIV-1 LTR-driven vectors that contained different export elements or no export element at all. The RNA expressed from these vectors was complementary to the HIV-1 env region and included either a Rev Response Element (RRE), which used the Rev/RRE export pathway, or a MPMV Constitutive Transport Element (CTE), which used the Tap/Nxf1 export pathway. The RRE-driven antisense RNA efficiently inhibited p24 production from an RREdriven provirus, whereas the CTE-driven antisense inhibited only at higher concentrations. The vector without the export element failed to inhibit. The RREdriven antisense also efficiently inhibited p24 production from a pNL4-3 provirus that uses the CTE for RNA export, indicating that cotargeting was not essential for efficient antisense inhibition. On the other hand, the CTE-driven antisense demonstrated a greater efficiency of antisense inhibition on the CTE-driven provirus than it did on the RRE-driven provirus, although in both cases the CTEdriven antisense was not as efficient as the RRE-driven antisense. Thus, efficient antisense inhibition required that the antisense RNA trafficked through 2 the Rev/RRE pathway. When RevM10-Tap and Nxt1 were coexpressed with RRE-driven provirus and antisense, forcing both the target and antisense RNA to use the Tap pathway, p24 production inhibited less efficiently. In fact, the RREdriven antisense construct inhibited p24 production at similar levels as the CTEdriven construct. Mechanistic studies demonstrated that nuclear retention does not contribute to antisense inhibition, since the GagPol and antisense RNA localized to the cytoplasmic fraction. We examined the stability of vector and GagPol target RNA to determine if degradation contributed to antisense inhibition and found that netiher vector nor target RNA was rapidly degraded. Since GagPol RNA efficiently localized to the cytoplasm and was not degraded in the presence of antisense RNA, we wanted to distinguish between an effect on protein levels and an effect on particle assembly and release. To do this, we examined protein expression levels from a provirus that produced GagPol Pr160 that could not be assembled into viral particles or released. We found that coexpression of the RRE-driven antisense led to reduced GagPol Pr160 levels, suggesting that the antisense RNA did not affect virus assembly or release, but rather protein levels. To determine if antisense expression had non-specific effects on protein expression, we assayed HIV-1 Nef expression from an RRE-driven provirus in the presence of antisense RNA. Nef is encoded by a multiply spliced HIV-1 RNA that does not contain the antisense target sequence, so specific antisense RNA inhibition would not be expected to reduce Nef levels. In the presence of RRE- 3 and CTE-driven antisense, Nef protein levels were not significantly reduced, demonstrating that antisense inhibition is specific to RNA containing the target sequence. Actively translated RNAs typically localize to the polyribosome. We examined localization of the GagPol RNA to the polyribosome in the presence of RRE-driven sense or antisense RNA. We found that there was no significant difference in polyribosomal localization of GagPol RNA in the presence of sense or antisense RNA. In addition, we determined that localization of the GagPol RNA was specific to the polyribosome, since EDTA treatment abolished localization. Our results demonstrate that antisense RNA expression leads to reduced GagPol protein levels but not reduced localization to the polyribosome. In addition, efficient antisense inhibition requires export of the antisense RNA via the Rev/RRE pathway.

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PHD (Doctor of Philosophy)
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