Uncoupling Human Immunodeficiency Virus Type 1
            <i>gag</i>
            and
            <i>pol</i>
            Reading Frames: Role of the Transframe Protein p6* in Viral Replication

b HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The Nterminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two Cterminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.

Section: Production and Functional Characterization Of Hiv-1 Variantsmentioning

confidence: 81%

Section: Production and Functional Characterization Of Hiv-1 Variantsmentioning

confidence: 99%

Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

Marco

Heuser

Glass

et al. 2012

“…The p1 peptide sequence is thought to be nonessential as long as it is proteolytically cleaved, because the majority of it can be deleted without significantly impacting infectivity (37). The mutations also alter the sequence of p6* in Gag-Pol, which overlaps p1-p6 and is also widely dispensable for infectivity (38). The M4 and M5 mutants have altered p1 peptide sequences and were nearly as infectious as the WT (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

García‐Miranda

Becker

Benner

et al. 2016

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...

“…The plasmid pNL4-3 unc , uncoupling the gag and pol open reading frames (ORFs) of HIV-1, was constructed according to a previous report (34). A silent unique MluI restriction site was introduced into pNL4-3 at nucleotide position 1356 with respect to the start of the gag reading frame.…”

Section: Methodsmentioning

confidence: 99%

“…Mutations in p6 also affect the sequence of the overlapping p6* pol region and may, in addition, influence frameshifting efficiency, thereby confounding the interpretation of results. To address this problem, we made use of a recently described HIV-1 derivative in which the gag and pol reading frames are uncoupled by mutagenesis of the authentic frameshift signal in conjunction with a short sequence duplication introducing a functional frameshift signal directly upstream of the gag stop codon (34). In this context, mutations within p6 do not affect the primary sequence of Pol.…”

Section: Prediction Of Potentially Phosphorylated Amino Acids In Hiv-mentioning

confidence: 99%

Comprehensive Mutational Analysis Reveals p6 ^Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

Radestock

Morales

Rahman

et al. 2013

bThe structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.