Use of modified U1 snRNAs to inhibit HIV-1 replication

Abstract

Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 3' polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate assembly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti-viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the constructs suggests that this strategy has significant potential as an HIV therapeutic.

Figure 1

U1 snRNA variants targeted to HIV-1 are able to suppress HIV-1 gene expression. (A) Outline of HIV-1 genome structure. Sites targeted by U1 snRNA variants are indicated by numbered lines above and below reading frames. (B) Sequence logos showing the conservation of sequences targeted by five U1 snRNA variants. Selected 10 nt sequences identified by MSA are boxed, the height of the individual base reflecting the degree of conservation at that position among HIV-1 strains. (C) Effect of anti-HIV-1 U1 snRNAs on viral protein expression. Cells were transfected with a plasmid containing HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-), CMV PLAP and one of the indicated U1 expression constructs. Forty-eight hours post-transfection, cells were harvested and level of p24 production assessed by western blot. Effect on secreted alkaline phosphatase (SEAP) expression (D) was performed in parallel as outlined in Materials and Methods and data are shown.

Figure 2

Suppression of HIV-1 gene expression occurs at the level of both viral protein and RNA. Cells were transfected with proviral DNA (pHxb2 R-/RI-) and 4 μg of U1 wt/αCAT expression vector or increasing amounts of αHIV #1/#5 (0.25, 0.5, 1, 2 or 4 μg) alone or in combination. Forty-eight hours post-transfection, cells were harvested and either (A) used to examine HIV-1 p24 expression or (B) RNA extracted and levels of RNA determined by northern blot. Representative gels are shown indicating the position of the viral RNAs (upper), GAPDH (middle) and U1 snRNA variants (lower).

Figure 3

HIV-1 Suppression is dependent upon the integrity of the U1 snRNP. (A) Schematic of U1 snRNA structure with the location and nature of U1 mutants indicated. Shown are the regions of α-HIV U1 #1 and #5 that were mutated to identify the protein components of the snRNP required to suppress viral gene expression. Mutations were as follows: deletion of stem–loop 1 (ΔSL1), deletion of stem–loop 2 (ΔSL2), substitutions in stem–loop 1 (SL1mut), substitutions in the loop region of stem–loop 2 (SL2mut) and mutations in the Sm binding region that abolishes assembly of the Sm core complex (ΔSm). (B and C) Effect of anti-HIV-1 #5/#1 mutation on Suppression of HIV-1 Gene Expression Cells were transfected with a plasmid containing HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-) and mutants of anti-HIV #5 (B) or #1 (C). Forty-eight hours post-transfection, cells were harvested and level of p24 production assessed by western blot.

Figure 4

SL2mut variants of anti-HIV U1 snRNAs retain anti-viral activity. (A) Cells were transfected with HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-) and vector expressing anti-HIV #1/#5 SL2mut snRNA either alone or in combination. Forty-eight hours post-transfection, cells were harvested and lysates analyzed for p24 expression by western blot. (B) Cells were transfected with HIV-1 proviral DNA (HIV-1 Hxb2 R-/RI-) and vector expressing anti-HIV #1/#5 wild-type, SL2mut, or SL1mut snRNAs either alone or in combination. Total RNA was subsequently extracted and levels of viral RNA, and GAPDH RNA assayed by northern blot. Shown is representative gel of the results obtained. Identity of viral RNAs, GAPDH RNA and U1 snRNA variants (lower) are as indicated.

Figure 5

Cells stably expressing anti-HIV #5 U1 snRNA show reduced capacity to support HIV-1 replication. HeLa CD4⁺ cells lines were isolated following transfection with pBABE vectors expressing without insert (puro), U1wt, or anti-HIV #5. Following selection for stable integrants, the pooled clones were challenged with HIV-1 and p24 production assayed five days post-infection by ELISA. Shown are the averages of two independent experiments and a total of four independent assays. Asterisk indicates values that are different from control cells (Mock) at a P-value <0.05.