Abstract:BackgroundHIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and −2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants.ResultsWhereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core,… Show more
“…Particle release formation was comparable, as indicated by ELISA ( panel D ). As reported previously, the substitution of Ala restored processing of CA-SP1 almost completely [22]. Since the substitution of Ala alters the PR, we can conclude that the altered PR can cleave CA-SP1, permitting virus maturation, and is responsible for the maintaining of the infectivity in the S40A variant.Fig.…”
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0298-1) contains supplementary material, which is available to authorized users.
“…Particle release formation was comparable, as indicated by ELISA ( panel D ). As reported previously, the substitution of Ala restored processing of CA-SP1 almost completely [22]. Since the substitution of Ala alters the PR, we can conclude that the altered PR can cleave CA-SP1, permitting virus maturation, and is responsible for the maintaining of the infectivity in the S40A variant.Fig.…”
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0298-1) contains supplementary material, which is available to authorized users.
“…Of these, desmoglein-2 interacts with intermediate filaments, while protein 4.1, cortactin, afadin, Filamin B, the CD2-associated protein (CD2AP), and band 4.1-like protein 2 all have been implicated as involved in actin cytoskeleton arrangements (26,68,72,84); this presumably underscores the importance of actin filament and membrane reorganization during virus assembly and potentially identifies targets of virus assembly interference. It also may pertain to the regulation of budding, since cortactin and CD2AP bind ESCRT-associated proteins Alix and TSG101 (26,86). One of the remaining two proteins in the cytoskeletal group, AHNAK, is recruited to cholesterol-rich domains at the PM by annexin 2, which plays a cell type-dependent role in the production of infectious HIV-1 (87,88).…”
Section: Discussionmentioning
confidence: 99%
“…In particular, the destinations of viral and cellular RNAs include actively translating ribosomes; stress granules (SGs), where mRNAs on stalled preinitiation complexes are stored or sorted; and processing bodies (PBs), involved in RNA silencing and decay (22,(90)(91)(92)(93)(94)(95). Previously, HIV-1 Gag proteins were found to associate with elongation factor 1 alpha, via RNA bridges to MA and NC, and this interaction was proposed to induce a shift from Gag protein translation to vRNA encapsidation (26). PrGag also was shown to associate with the Staufen-1 SG protein (22,90,91), which may foster both virus assembly and vRNA encapsidation.…”
Section: Discussionmentioning
confidence: 99%
“…Interestingly, over half of the biotinylated proteins have been (Table 2, far right column) (22,26,35,36,42,50,(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)85). As can be seen, the list includes a variety of translation factors, cytoskeleton-associated proteins, membrane proteins, and RNA-processing proteins: these are discussed in more detail below.…”
Section: Biotinylated Proteins In Virus Particlesmentioning
confidence: 99%
“…During transit to the PM, PrGag associates with full-length HIV-1 viral RNAs (vRNAs), which is controlled, in part, by virtue of a preferential binding of NC domains to the vRNA encapsidation (E) or Psi (⌿) element (14)(15)(16)(17)(18)(19)(20). Delivery of PrGag proteins and vRNAs to the PM would appear to require interaction with and reconfiguration of the cellular cytoskeleton, including the cortical actin network, but details regarding these interactions are scant (21)(22)(23)(24)(25)(26). However, once at the PM, major PrGag-PrGag contacts appear to be mediated by CA domains, while budding of membrane-coated virus particles is facilitated by p6 recruitment of cellular endosomal sorting complexes required for transport (ESCRT) and ESCRT-associated factors (20,(27)(28)(29)(30)(31).…”
We have examined the interactions of wild-type (WT) and matrix protein-deleted (⌬MA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ⌬MA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ⌬MA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference.
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