The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BackgroundThe p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy.ResultsRemarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV.ConclusionsOur results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor

Characterization of HIV-1 protease autoprocessing trans-cleavage mechanism

2014 Spring.Includes bibliographical references.HIV protease is an aspartic acid enzyme responsible for the cleavage reactions essential in the maturation (infectivity) of the viral particle. Protease inhibitors (non-cleavable substrate analogs) have been potent tools in combating HIV infection as well as its result - AIDS. However, the emergence of drug-resistant viruses in patients treated with these inhibitors is an ongoing concern. Thus there is a growing need to find additional therapeutic targets and treatments to supplement the currently available protease inhibitors. A promising new target for drug development is protease autoprocessing which is a virus-specific process responsible for the release of the mature protease from its precursor (Gag-Pol). Unfortunately, structural and mechanistic information pertaining to autoprocessing are yet insufficient. According to the mature protease structure, it is speculated that precursor dimerization is essential for autoprocessing to occur. We have developed a model system to specifically examine the trans-cleavage mechanism mediated by engineered fusion precursors (differentially labeled substrate and enzyme, respectively). Using this system, we demonstrate that trans-cleavage happens between fusion precursors both in the presence and absence of a dimer inducing fusion tag (DIFT). Trans-cleavage was also observed when monomeric fusion tags were attached to the fusion precursor. These results hint that autoprocessing mediated by the fusion precursor is independent of dimer-inducing tag in our model system

Sequence features governing aggregation or degradation of prion-like proteins

<div><p>Enhanced protein aggregation and/or impaired clearance of aggregates can lead to neurodegenerative disorders such as Alzheimer’s Disease, Huntington’s Disease, and prion diseases. Therefore, many protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Prions, which generally result from self-propagating protein aggregates, must therefore evade or outcompete these quality control systems in order to form and propagate in a cellular context. We developed a genetic screen in yeast that allowed us to explore the sequence features that promote degradation versus aggregation of a model glutamine/asparagine (Q/N)-rich prion domain from the yeast prion protein, Sup35, and two model glycine (G)-rich prion-like domains from the human proteins hnRNPA1 and hnRNPA2. Unexpectedly, we found that aggregation propensity and degradation propensity could be uncoupled in multiple ways. First, only a subset of classically aggregation-promoting amino acids elicited a strong degradation response in the G-rich prion-like domains. Specifically, large aliphatic residues enhanced degradation of the prion-like domains, whereas aromatic residues promoted prion aggregation without enhancing degradation. Second, the degradation-promoting effect of aliphatic residues was suppressed in the context of the Q/N-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation. Degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated activity for these residues in yeast prion domains. Collectively, these results provide key insights into how certain aggregation-prone proteins may evade protein quality control degradation systems.</p></div

Degradation of the A2 PrLD and stability of the Sup35 ND upon insertion of hydrophobic residues do not depend on the Sup35 oligopeptide repeat domain (ORD), M-domain, or C-domain.

<p>Progressively increasing hydrophobic content in the A2 PrLD (A) when fused to GFP alone (<i>top</i>) or with the remainder of the Sup35NM domains and tandem FLAG tags (<i>bottom</i>) enhances degradation rate of the A2 PrLD fusion proteins. By contrast, insertion of hydrophobic residues in the Sup35 ND (B) fused to GFP (<i>top</i>) or Sup35NM-2xFLAG (<i>bottom</i>) does not decrease stability of the Sup35 ND fusion proteins. Data represent means ± SDs (n = 3).</p

Degradation of the A2 PrLD and prion aggregation of Sup35 occur at similar hydrophobic content thresholds.

<p>(A) Two or more hydrophobic residues inserted into the A2 PrLD resulted in a robust <i>ADE</i>^<i>+</i> phenotype and accelerated degradation of the A2 PrLD. (B) Insertion of three or more hydrophobic residues into Sup35 led to a progressive increase in the frequency of white sectors on YPD without affecting Sup35 turnover. (C) To quantify the frequency of <i>ADE</i>^<i>+</i> colony formation, serial dilutions of cells expressing each A2-Sup35 fusion were plated onto SC-ade. Degradation of the A2 PrLD upon insertion of two or more hydrophobic residues was correlated with a binary-like switch from <i>ade</i>^- to <i>ADE</i>⁺. (D) <i>ADE</i>⁺ isolates from the A2 PrLD mutants were not curable by GuHCl. To test for curability of the <i>ADE</i>⁺ phenotype, individual <i>ADE</i>⁺ colonies were streaked on YPD (-) or YPD plus 4mM GuHCl (+), and then re-streaked onto YPD to test for loss of the <i>ADE</i>⁺ phenotype. (E) Insertion of three or more hydrophobic residues in the Sup35 ND leads to a progressive increase in <i>ADE</i>⁺ growth. (F) <i>ADE</i>⁺ isolates from the Sup35 mutants were curable by GuHCl, consistent with the <i>ADE</i>^<i>+</i> phenotype resulting from prion formation. (G) Insertion of hydrophobic amino acids at other positions in the A2 PrLD also promoted protein degradation. (H) Insertion of hydrophobic amino acids at other positions in the Sup35 nucleation domain had little or no effect on protein turnover.</p

Yeast prion domains are enriched in amino acids that are prion-prone but not degradation-promoting.

<p>Average degradation scores from the A1 PrLD and A2 PrLD libraries are plotted against yeast prion propensity scores for individual amino acids (A) or amino acid groups (B). Within native yeast prion domains, commonly occurring amino acids (Q, N, and aromatic amino acids) exhibit a combination of high prion propensity and low degradation propensity. Colors correspond to the amino acid groups in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007517#pgen.1007517.t001" target="_blank">Table 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007517#pgen.1007517.g003" target="_blank">Fig 3</a>.</p

Amino acid representation among <i>ADE</i>⁺ and <i>ade</i>^- isolates.

<p>Amino acid representation among <i>ADE</i>⁺ and <i>ade</i>^- isolates.</p

High Q/N content of Sup35 antagonizes degradation.

<p>(A) Sequences of the partial or full Q/N/G substitution constructs. Glycines are indicated in red, while glutamines and asparagines are in green. (B) Partial or full substitution of Q/N residues for G within the Sup35 nucleation domain resulted in the <i>ADE</i>^<i>+</i> degradation phenotype (<i>left</i>), and a step-wise increase in degradation rate (<i>right</i>). Partial substitution of G residues for Q/N residues within the A2 PrLD resulted in an <i>ade</i>^<i>-</i> phenotype (<i>left</i>), and corresponding decrease in degradation rate (<i>right</i>). Full substitution of remaining G residues for QN residues had mixed effects.</p

Amino acid degradation scores are sufficient to identify degradation-promoting and inhibiting peptides from an independent peptide library.

<p>(A) Predicted degradation-promoting (LVIAGDIS, YISVYVAG, and LYVITNFI) or inhibiting (SRGDRSSG and GIRRDCGC) peptides were substituted into the A2 PrLD (numbers in parentheses indicate the sum of the individual amino acid scores derived from the A2 PrLD degradation library). Predicted degradation-promoting peptides led to an <i>ADE</i>^<i>+</i> phenotype and accelerated degradation of the A2 PrLD. Predicted degradation-inhibiting peptides led to the <i>ade</i>^<i>-</i> and showed no increase in degradation rate of the A2-Sup35 fusion. (B) In the context of the Sup35 ND, all peptides were stable over 5 hrs and conferred a predominantly <i>ade</i>^<i>-</i> phenotype.</p

The A2-Sup35 protein exhibits PrLD-dependent prion activity.

<p>(A) A2-Sup35 prion maintenance requires continual expression of the A2-Sup35 prion domain. A covering plasmid expressing a copy of Sup35 lacking the prion domain was shuffled into a [<i>PRION</i>⁺] isolate (A2 D290V). In the [<i>PRION</i>⁺] strain, cells became <i>ade</i>^- upon loss of the A2-Sup35 plasmid, and remained <i>ade</i>^- when the plasmid was re-introduced. (B) A2-Sup35 prions are unable to convert wild-type Sup35. Co-expression of wild-type Sup35 in the [<i>PRION</i>⁺] strain resulted in a reversion to the <i>ade</i>^- phenotype that was maintained upon loss of the A2-Sup35 plasmid, suggesting that the A2-Sup35 prion form is not efficiently transmitted to wild-type Sup35.</p