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Tamara Bar-Magen Richard D. Sloan Daniel A. Donahue Bj?rn D. Kuhl Alexandra Zabeida Hongtao Xu Maureen Oliveira Daria J. Hazuda Mark A. Wainberg 《Journal of virology》2010,84(18):9210-9216
MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender cross-resistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9, 26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or EVG. MK-2048 (28) is a prototype second-generation INSTI that retains potency against viruses containing common single and double mutations observed in the clinic with first-generation agents with a 95% inhibitory concentration (IC95) in the nM range. MK-2048 has been previously reported to be active against viruses resistant to RAL and EVG (28, 29). Given common mechanisms of action among INSTIs and a lack of structural information on integrase inhibitor complexes with resistance mutations, an understanding of resistance to second-generation agents such as MK-2048 is important.This article describes the selection of resistance to MK-2048 in tissue culture and the characterization of mutations associated with such resistance, G118R and E138K. The identification of distinct mutations which appear to confer resistance to MK-2048 and not to either RAL or EVG has potential implications for understanding the structural basis for the second-generation profile of this compound as well as future drug discovery and development efforts focused on this mechanism. 相似文献
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Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain. 相似文献
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Nicole M. Walley Boris Julg Samuel P. Dickson Jacques Fellay Dongliang Ge Bruce D. Walker Mary Carrington Myron S. Cohen Paul I.W. de Bakker David B. Goldstein Kevin V. Shianna Barton F. Haynes Norman L. Letvin Andrew J. McMichael Nelson L. Michael Amy C. Weintrob 《Cell host & microbe》2009,5(5):408-410
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Background
Tetherin/BST-2 is a recently-identified potent restriction factor in human cells that restricts HIV particle release following particle formation and budding at the plasma membrane. Vpu counteracts tetherin''s restriction of particle release in a manner that has not yet been fully defined. We recently identified calcium-modulating cyclophilin ligand (CAML) as a Vpu-interacting protein that also restricts particle release. We hypothesized that CAML may act to enhance tetherin-mediated restriction of particle release and thereby explain how two distinct factors could be responsible for Vpu-responsive restriction.Methodology/Principal Findings
Endogenous levels of tetherin in human cells correlated well with their restriction pattern and responsiveness to Vpu, while levels of cellular CAML protein did not. Tetherin but not CAML was inducible by interferon in a wide variety of human cells. Stable depletion of human CAML in restrictive HeLa cells had no effect on cell surface levels of tetherin, and failed to relieve tetherin-mediated restriction. Stable depletion of tetherin from HeLa cells, in contrast, rendered HeLa cells permissive and Vpu-unresponsive. Tetherin but not CAML expression in permissive human cells rendered them restrictive and Vpu responsive. Depletion of CAML had no influence on cell surface levels of tetherin.Conclusions/Significance
We conclude that tetherin restricts particle release and does not require CAML for this effect. Furthermore, these results do not support a major role for CAML in restricting HIV particle release in human cells. 相似文献13.
Claire E. Le Pichon Sara L. Dominguez Hilda Solanoy Hai Ngu Nicholas Lewin-Koh Mark Chen Jeffrey Eastham-Anderson Ryan Watts Kimberly Scearce-Levie 《PloS one》2013,8(4)
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive paralysis due to motor neuron death. Several lines of published evidence suggested that inhibition of epidermal growth factor receptor (EGFR) signaling might protect neurons from degeneration. To test this hypothesis in vivo, we treated the SOD1 transgenic mouse model of ALS with erlotinib, an EGFR inhibitor clinically approved for oncology indications. Although erlotinib failed to extend ALS mouse survival it did provide a modest but significant delay in the onset of multiple behavioral measures of disease progression. However, given the lack of protection of motor neuron synapses and the lack of survival extension, the small benefits observed after erlotinib treatment appear purely symptomatic, with no modification of disease course. 相似文献
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Lei Feng Amit Sharma Alison Slaughter Nivedita Jena Yasuhiro Koh Nikolozi Shkriabai Ross C. Larue Pratiq A. Patel Hiroaki Mitsuya Jacques J. Kessl Alan Engelman James R. Fuchs Mamuka Kvaratskhelia 《The Journal of biological chemistry》2013,288(22):15813-15820
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance. 相似文献
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Siarhei Kharytonchyk Joshua D. Brown Krista Stilger Saif Yasin Aishwarya S. Iyer John Collins Michael F. Summers Alice Telesnitsky 《Journal of molecular biology》2018,430(14):2066-2079
The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5′ leader Ψ elements plus poorly defined additional features. We previously defined minimal 5′ leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5′ leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5′ leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination. 相似文献
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The Proteolytic Cleavage of Human Immunodeficiency Virus Type 1 Nef Does Not Correlate with Its Ability To Stimulate Virion Infectivity 总被引:7,自引:5,他引:2
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The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan57 decreased the efficiency of Nef processing, while mutating leucine58 had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a nef-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity. 相似文献