Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

Abstract

Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus-or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gagpolymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCEHIV, like many retroviruses, utilizes a ؊1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies. The genome of human immunodeficiency virus type 1 (HIV-1), like that of other retroviruses, has three genes that encode the structural proteins Gag, polymerase (Pol), and Env. The expression of the gag gene results in the synthesis of the Gag precursor protein, p55, which is subsequently processed by the viral protease to release the mature Gag proteins p17 (matrix protein), p24 (capsid), p15 (nucleocapsid), and p6 (late domain) and two so-called spacer peptides (SP) that flank p15, namely, p2 (SP1) and p1 (SP2), respectively (1). The synthesis of Gag precursor protein alone is sufficient for the assembly and release of noninfectious virus-like particles (VLPs) (2). The pol gene codes for the p160 polyprotein, which is su...

Modulation of HIV-1 Gag/Gag-Pol frameshifting by tRNA abundance

“…With native SS1, the joint activity of SS1 and pSS2 is not different from SS1 alone, but when SS1 is mutated to a non-shifty sequence, the C 5 U mutation in the pSS2 is sufficient to support a level of -1FS that may be sufficient for virus propagation. The finding that pSS2 can alleviate the detrimental effects of SS1 mutations is consistent with previous in vitro and in vivo reports (26)(27)(28)(29)(30). Interestingly, during antiviral therapy, drug-resistant herpes simplex viruses also develop an unusual slippery site that supports both -1 and +1FS at levels sufficient for virus replication and pathogenicity despite the treatment (81,82).…”

“…Further HIV frameshifting product heterogeneity may result from -2 frameshifting (25). In addition, the gag-pol gene has a second, putative slippery site (pSS2) 38 nt downstream of the canonical SS1 (26)(27)(28)(29)(30). This slippery site is also conserved albeit to a lesser degree than the first slippery site (31).…”

“…This slippery site is also conserved albeit to a lesser degree than the first slippery site (31). The sequence of pSS2 (U 1 UUU 4 CUU 7 ) is not particularly slippery, but a substitution of C 5 with U (C 5 U), which appears as a compensatory resistance mutation during anti-HIV therapy, may facilitate additional FS at this otherwise silent site (26)(27)(28)30).…”

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

“…For thin-section EM, HEK293T cells were cultured in six-well dishes, transfected as described above, and processed as previously described (137). At 48 h posttransfection, cells were fixed in a solution of 2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4, for ϳ2 h at room temperature.…”

Mechanisms and biomedical implications of –1 programmed ribosome frameshifting on viral and bacterial mRNAs

“…The mutation also turns the U 1 UUU 4 CUU 7 sequence into U 1 UUU 4 UUU 7 , which can support ribosome slippage (Fig. 1A) [97][98][99][100][101]. The mutated slippery pSS2 supports both -1 and -2 slippage, but its contribution to the overall frameshifting efficiency depends on the availability of SS1 ( Fig.…”