Trafficking through the Rev/RRE Pathway Is Essential for Efficient Inhibition of Human Immunodeficiency Virus Type 1 by an Antisense RNA Derived from the Envelope Gene

Abstract

A human immunodeficiency virus type 1 (HIV-1)-based vector expressing an antisense RNA directed against HIV-1 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 unmodified antisense RNAs has generally been disappointing. To further analyze the basis for this, we examined the effects of different plasmid-based HIV-1 long-terminalrepeat-driven constructs expressing antisense RNA to the same target region in HIV-1 but containing different export elements. Two of these vectors were designed to express antisense RNA containing either a Rev response element (RRE) or a Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). In the third vector, no specific transport element was provided. Efficient inhibition of HIV-1 virus production was obtained with the RRE-driven antisense RNA. This construct also efficiently inhibited p24 production from a pNL4-3 provirus that used the MPMV CTE for RNA export. In contrast, little inhibition was observed with the constructs lacking an RRE. Furthermore, when the RRE-driven antisense RNA was redirected to the Tap/Nxf1 pathway, utilized by the MPMV CTE, through the expression of a RevM10-Tap fusion protein, the efficiency of antisense inhibition was greatly reduced. These results indicate that efficient inhibition requires trafficking of the antisense RNA through the Rev/RRE pathway. Mechanistic studies indicated that the Rev/RRE-mediated inhibition did not involve either nuclear retention or degradation of target mRNA, since target RNA was found to export and associate normally with polyribosomes. However, protein levels were significantly reduced. Taken together, our results suggest a new mechanism for antisense inhibition of HIV mediated by Rev/RRE.

HIV, ADAR Editing, The Rev-RRE Pathway and Antisense RNA-mediated Inhibition

“…In this and the previous study by Ward et al it has also been clearly demonstrated that efficient AS RNA-mediated inhibition of HIV-1 replication is Rev/RRE pathway dependent 127 . In addition, our data have demonstrated that presence of a panhandle in RRE-driven AS env RNA significantly promotes AS inhibition.…”

“…In the construction of the HIV vectors that were used in the Ward et al study 127 ( Figure 22), a region that was complementary to part of the envelope To directly compare the inhibition that could be achieved with these vectors, constructs with and without panhandles were co-transfected into 293T cells together with the pNL4-3 proviral plasmid and inhibition was measured as a reduction in p24 levels, as previously described 127 . The result from the analysis showed that RRE-driven antisense vector with a panhandle was about 10-fold more effective in inhibiting HIV than the corresponding vector lacking a panhandle at a molar ratio of 1:1 ( Figure 25A).…”

“…A principle of antisense-mediated inhibition of gene expression is that trafficking through the HIV-1 Rev/RRE pathway 127 . As described above, the HIV-…”

“…2009 127 , we discovered that the constructs used in that study expressed an AS RNA which contained a 287 bp dsRNA panhandle formed by sequences in the env region that creates an inverted repeat. This panhandle was not present in the original vector used in the Lu et al 2004 experiments 126 .…”

“…Rev/RRE pathway 127 . The HIV-1 Rev/RRE pathway is best known for its ability to overcome nuclear retention of HIV-1 unspliced and incompletely spliced transcripts and mediate their nuclear export 36,38,39 .…”