The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles

Abstract

Background: The process of HIV-1 genomic RNA (gRNA) encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements in the canonical packaging signal (ψ). Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site) from the 5' untranslated region (UTR). Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV) vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles.

Results: We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ). Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons); however, the cDNA forms are episomes predominantly in the 1-LTR circle form.

Conclusions: Premised on encapsidation of a heterologous RNA into HIV-1 viral particles, our findings define a functional HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation.

Figure 1

HIV-1 Rev/RRE system and cis elements in the 5'UTR augment vector titers. A. Full-length MLV/HIV chimeric vector RNAs are expressed from a CMV (cytomegalovirus) promoter in transfected 293T cells. MLV and HIV cis elements can be distinguished by black underscore. Chimeric vector names are represented as MLV/HIV followed by corresponding HIV cis elements incorporated: RRE (Rev Response Element), R (repeat), U5 (unique region 5), PS (packaging signal comprised of ψ [canonical packaging signal and into 5' Gag region]), cPPT (central polypurine tract), PBS (primer binding site). Also incorporated are the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), FLuc (firefly luciferase gene), and GFP (green fluorescent protein gene). HIV-1 Gag-Pol 4X CTE helper construct was used to express structural and enzymatic proteins to generate viral particles independent of HIV-1 Rev protein. B. Vector titers normalized to p24 are shown in the absence (white bars) and presence (black bars) of Rev. The influence of adding HIV-1 cis elements to the MLV vector is indicated by fold increases in the presence of Rev relative to the standard MLV vector. Fold increases for MLV/HIV RRE + PS (38 fold) and MLV/HIV RRE + RU5 (5 fold) are not indicated on the graph. C. Luciferase levels normalized to total protein are shown for each vector. D. Titers expressed as a ratio to luciferase are shown as arbitrary units (AU). Fold increases for MLV/HIV RRE + PS (22 fold) and MLV/HIV RRE + RU5 (4 fold) are not indicated on the graph. Error for all bar graphs is expressed as ±S.D. All experiments were performed in triplicate.

Figure 2

Transduction of 293T cells with chimeric MLV/HIV vectors packaged into HIV-1 viral particles. A and B. 293T cells were transduced with equivalent amounts of p24 capsid protein (50 ng), as determined for each of the indicated chimeric vectors. The influence of the HIV-1 Rev/RRE system, and 5' UTR cis elements, on transduction was assessed by fluorescence microscopy (A) and FACscan analysis (B) at 7 days post-transduction. C. 293T cells were transduced in the absence (No RT Inhibitor), or presence (+RT Inhibitor), of the HIV-1 specific non-nucleoside reverse transcriptase inhibitor, etravirine (100 nM). Transduced cells were assessed by fluorescence microscopy and FACscan analysis. D. The capacity of the MLV/HIV RRE + RU5PS vector to be stably maintained after 4 cell passages was examined by FACscan analysis. The percent GFP positive cells are indicated for each FACscan and 293T negative control (NC) cells are shown.

Figure 3

HIV-1 Rev/RRE and cis elements in the 5'UTR cooperatively enhance RNA encapsidation into HIV-1 viral particles. A. Vector RNA was measured by qRT-PCR and expressed in arbitrary units (AU). RNA levels for all graphs are shown in the absence (white bars) and presence (black bars) of Rev. The influence of adding HIV-1 cis elements to the MLV vector is indicated by fold increases in the presence of Rev relative to the standard MLV vector. Fold increases in vector RNA for MLV/HIV RU5PS (1.9 fold), MLV/HIV RRE + PS (17 fold) and MLV/HIV RRE + RU5 (7.7 fold) are not indicated on the graph. B. Cytoplasmic RNA was isolated from vector producer 293T cells at the time of vector harvesting. Relative RNA levels were obtained and recorded as done for vector RNA in part A. C. Efficiency of encapsidating RNA into HIV-1 viral particles is expressed as a ratio of vector RNA in viral particles to cytoplasmic RNA available for encapsidation. Relative levels are expressed like vector RNA in part A. Fold increases for MLV/HIV RU5PS (1.2 fold), MLV/HIV RRE + PS (6.7 fold) and MLV/HIV RRE + RU5 (3.9 fold) are not indicated on the graph. D. Northern blot analysis of cytoplasmic and vector RNA isolated from MLV and MLV/HIV RRE in the absence (-) and presence (+) of Rev. Vector length RNA species were detected with a GFP labeled probe, as well as an additional RNA species (labeled GFP) generated from the internal CMV promoter. E. Northern blot analysis of cytoplasmic and vector RNA isolated from MLV/HIV RU5PS and MLV/HIV RRE + RU5PS in the absence (-) and presence (+) of Rev. Vector length RNA species were detected with a probe to a region in the 5' end of the vector, as well as an additional RNA species (labeled 'partial vector RNA'). Cytoplasmic and vector RNAs are shown at different exposures of the same blot. Last lane (far right) is a shorter exposure of adjacent left lane. Error for all bar graphs is expressed as ±S.D. All experiments were performed in triplicate.

Figure 4

HIV-1 Rev/RRE and cis elements in the 5'UTR do not influence vector titers after packaging into MLV viral particles. A. Titers of MLV/HIV chimeric vectors were obtained by scoring for GFP positive cells following transduction of 293T cells. Titers are expressed as transducing units (TU) normalized to the amount of RT units (counts per minute [CPM]). B. Normalized luciferase levels were determined in transfected 293T producer cells. Luciferase levels were normalized to total cell protein. C. Titers (part A) expressed as a ratio to levels of luciferase (part B) shown in arbitrary units (AU). All experiments were executed in the absence (white bars) and presence (black bars) of Rev. Error for all bar graphs is expressed as ±S.D. All experiments were performed in triplicate. D and E. 293T cells were transduced with equivalent amounts of RT units (6 × 10⁵ CPM), as determined for each of the indicated chimeric vectors. The influence of the HIV-1 Rev/RRE system, and 5' UTR cis elements, on transduction was assessed by fluorescence microscopy D and FACscan analysis E at 7 days post-transduction. The percent GFP positive cells are indicated for each FACscan.

Figure 5

HIV-1 Rev/RRE and cis elements in the 5' UTR do not augment RNA encapsidation into MLV viral particles. A. Vector RNA packaged into MLV derived viral particles was isolated from equivalent amounts of RT units in the media of 293T producer cells. RNA levels were measured by qRT-PCR and are expressed as arbitrary units (AU). RNA levels are shown in the absence (white bars) and presence (black bars) of Rev. B. Cytoplasmic RNA was isolated from vector producer cells coincident with harvesting vector particles. Relative levels are expressed similar to vector RNA in part A. C. Efficiency of encapsidating RNA into MLV viral particles is expressed as a ratio of vector RNA in viral particles to cytoplasmic RNA available for encapsidation. D. Transduction of 293T cells with MLV/HIV RRE + RU5PS at 5 days post-transduction (no passaging of cells, P0), and after 5 passages of cells (P5). Percent GFP positive cells were assessed by FACscan analysis and compared to non-transduced (No Vector) 293T cells. Error for all bar graphs is expressed as ±S.D. All experiments were performed in triplicate.

Figure 6

Heterologous MLV RNAs form, predominantly, 1-LTR episomal cDNAs following delivery with HIV-1 viral particles. MLV/HIV RRE + RU5PS chimeric vector was packaged into HIV-1 and MLV viral particles in the presence of Rev. 293T cells were transduced with equivalent transducing units for HIV-1 and MLV packaged vectors. Total cellular DNA was harvested at 5 days post-transduction (episomal and integrated vector DNA, P0), and after five cell passages (integrated vector DNA, P5). Vector DNA copy number as measured by qPCR to the WPRE A, and β-globin DNA copy number B, were determined by qPCR. Data are shown without cell passages (P0; white bars) and after 5 cell passages (P5; black bars). Vector DNA copy number was normalized to β-globin copy number C, and fold decreases in vector DNA levels, after passaging cells, are shown. Error for all bar graphs is expressed as ±S.D. D. Southern blot analysis of total DNA isolated from 293T cells transduced with MLV/HIV RRE + RU5PS vector packaged into either HIV, or MLV, viral particles (as indicated above lanes). Total DNA was isolated at 5 days posttransduction (P0) and after 5 passages of cells (P5). DNA was digested to distinguish between 2-LTR, 1-LTR, and linear episomal forms, as well as the vector backbone which is indicative of integrated vector DNA after passaging cells.

Figure 7

HIV-1 structural and enzymatic proteins can deliver a heterologously packaged RNA to non-dividing cells in vivo. A. MLV/HIV RRE chimeric vector used to investigate transduction of mouse neurons in vivo following packaging into MLV or HIV-1 viral particles. B. Mouse brains were injected into the striatum with equivalent transducing units of MLV/HIV RRE vector packaged into either MLV (left panels), or HIV-1 (right panels), derived viral particles. Brain sections were imaged by confocal microscopy following co-staining for neurons (NeuN, red) and vector particles (GFP, green). Images depicting both vector and neurons can be seen for MLV (top left and low magnification) and HIV (left top and low magnification) viral particles. Independent images of vector and neurons are shown for MLV (top middle and top right, respectively) and HIV (left middle and left bottom, respectively) viral particles. C. The graph represents the total number of cells scored for GFP only (white bars), and colocalized GFP + NeuN (black bars) from 5 mice in each group. Error for bar graph is expressed as ±S.D.