Matrin 3 is a co-factor for HIV-1 Rev in regulating post-transcriptional viral gene expression

Abstract

Post-transcriptional regulation of HIV-1 gene expression is mediated by interactions between viral transcripts and viral/cellular proteins. For HIV-1, post-transcriptional nuclear control allows for the export of intron-containing RNAs which are normally retained in the nucleus. Specific signals on the viral RNAs, such as instability sequences (INS) and Rev responsive element (RRE), are binding sites for viral and cellular factors that serve to regulate RNA-export. The HIV-1 encoded viral Rev protein binds to the RRE found on unspliced and incompletely spliced viral RNAs. Binding by Rev directs the export of these RNAs from the nucleus to the cytoplasm. Previously, Rev co-factors have been found to include cellular factors such as CRM1, DDX3, PIMT and others. In this work, the nuclear matrix protein Matrin 3 is shown to bind Rev/RRE-containing viral RNA. This binding interaction stabilizes unspliced and partially spliced HIV-1 transcripts leading to increased cytoplasmic expression of these viral RNAs.

Figure 1

Matrin 3 promotes the expression of Rev dependent RRE containing transcripts. A) HeLa cells were transfected with Matrin 3 and Tat along with HIV-1 LTR luciferase. Luciferase assays performed on cell lysates prepared from these cells did not show any effect of Matrin 3 on Tat dependent LTR transactivation. B) Matrin 3 enhances the expression of RRE containing RNA transcripts in the presence of Rev in HeLa cells. HeLa cells were transfected with 2.0 μg of Matrin 3 expression or control plasmid along with 0.5 μg of pCMV -GagPol-RRE plasmids in the presence or absence of Rev. HA-Matrin 3 significantly increased the expression of Gag from the reporter construct pCMV-GagPol-RRE in the presence of Rev (compare lanes 2 and 4). C) Gag expression from CTE containing pCMV-GagPol-CTE reporter was not effected by HA-Matrin 3 (compare lanes 5 and 6).

Figure 2

Matrin 3 increases HIV-1 production from transiently tranfected HeLa cells. A) HeLa cells were transfected with pNL4-3 along with WT Matrin 3, and the expression of viral proteins was analyzed on Western blots. Wild type HA-Matrin 3 (lane 2) enhanced viral protein expression. B) Matrin 3 knockdown using siRNA efficiently decreased cell endogenous Matrin 3 (lanes 2 and 3; top panel). Controls were scrambled irrelevant siRNAs. (lower two panels) HeLa cells were transfected with HIV-1 molecular clone pNL4-3 and either the control or the siRNA targeting Matrin 3. Western blot analysis of cell lysates showed that siRNA-mediated Matrin 3 knockdown reduced HIV-1 expression as indicated by decreased p24 expression (lanes 2 and 3). Loadings were normalized to β-actin.

Figure 3

Matrin 3 interacts with Rev in the presence of viral Rev-dependent RRE-containing RNA. A) Schematic representations of the RNAs expressed from the various p37Gag constructs. B) Co-immunoprecipitation of GFP-Rev occurs only in the context of p37-RRE. HeLa cells were transfected with either pCMV-HA (lanes 1-6), or pCMVHA-Matrin 3 (lane 7-12) and GFP-Rev (lanes 1-12) plasmids, along with the indicated versions of a p37Gag expression construct (see panel A and as indicated). Cell lysates were subjected to immunoprecipitation with anti-HA antibody. Western blot analysis of co-immunoprecipitations shows that interaction occurs between Rev and Matrin 3 in the presence of co-transfected p37RRE (lane 8, top panel) construct, but not p37, p37CTE, or codon optimized P37 Gag constructs that are Rev-independent (lanes 7 and 9-12, top panel). Lower two panels show the expression of Rev and Matrin 3 in cell lysates used for the immunoprecipitations, and the second panel from the top shows HA-Matrin 3 proteins recovered by the co-immunoprecipitations.

Figure 4

Matrin 3 RRMs are required for activity on HIV-1 RNA. A) Schematic representations of the RRM deletion mutants of Matrin 3. B) Western blot verification of the comparable expression of transfected Matrin 3 deletion mutants. Loadings were normalized to β-actin (bottom panel). C) Expression of wild type HA-Matrin 3 (lane 2), but not HA-Matrin 3 d264-595 (lane 3) nor HA-Matrin 3 d162-595 (lane 4), which lack the RRMs activated HIV-1 gene expression as measured by viral p55 or p24 levels.

Figure 5

Matrin 3 stabilizes RRE-containing RNA. A) (top) Schematic representations of the differently sized mRNA transcripts produced during HIV-1 replication. The 9 kb (unspliced) and 4 kb (singly spliced) viral transcripts contain the RRE cis-element and require Rev protein for expression. (bottom) HeLa cells were transfected with HIV-1 molecular clone pNL4-3 and either pCMV-HA or HA-Matrin 3 plasmids. Northern blot analysis of whole cell RNA demonstrated increased expression of unspliced 9 kb HIV-1 transcript (lane 2). Relative changes in the expression of 9 kb and 1.8 kb HIV-1 RNAs in cells, with and without Matrin 3, are shown by the numbers on the right. B) Matrin 3 increased the stability and promoted the nuclear export of HIV-1 unspliced RNA. HeLa cells were transfected with pNL4-3 with (red) or without (green) Matrin 3. RNA was isolated from whole cell lysates as well as nuclear and cytoplasmic fractions. qRT-PCR analysis of HIV-1 RNA was performed using primers specific for spliced and unspliced viral transcripts [29]. Transfection of Matrin 3 (red) resulted in modestly increased amounts of HIV-1 unspliced transcripts in the cells (top left panel, total), and a much larger increase in the distribution of unspliced HIV-1 transcripts into cytoplasm (top right panel, cytoplasmic). As control, Matrin 3 did not affect the stability or the distribution of GAPDH mRNA (bottom panels, GAPDH).