Publications by authors named "Alan Rein"

81 Publications

Across the Hall from Pioneers.

Authors:
Alan Rein

Viruses 2021 Mar 16;13(3). Epub 2021 Mar 16.

HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA.

I was fortunate to be associated with the lab of Stephen Oroszlan at the US National Cancer Institute from ~1982 until his conversion to Emeritus status in 1995. His lab made groundbreaking discoveries on retroviral proteins during that time, including many features that could not have been inferred or anticipated from straightforward sequence information. Building on the Oroszlan lab results, my colleagues and I demonstrated that the zinc fingers in nucleocapsid proteins play a crucial role in genomic RNA encapsidation; that the N-terminal myristylation of the Gag proteins of many retroviruses is important for their association with the plasma membrane before particle assembly is completed; and that gammaretroviruses initially synthesize their Env protein as an inactive precursor and then truncate the cytoplasmic tail of the transmembrane protein, activating Env fusogenicity, during virus maturation. We also elucidated several aspects of the mechanism of translational suppression in gene expression in gammaretroviruses; amazingly, this is a fundamentally different mechanism of suppression from that in most other retroviral genera.
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http://dx.doi.org/10.3390/v13030491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002223PMC
March 2021

Structural Mimicry Drives HIV-1 Rev-Mediated HERV-K Expression.

J Mol Biol 2020 12 14;432(24):166711. Epub 2020 Nov 14.

Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.

Expression of the Human Endogenous Retrovirus Type K (HERV-K), the youngest and most active HERV, has been associated with various cancers and neurodegenerative diseases. As in all retroviruses, a fraction of HERV-K transcripts is exported from the nucleus in unspliced or incompletely spliced forms to serve as templates for translation of viral proteins. In a fraction of HERV-K loci (Type 2 proviruses), nuclear export of the unspliced HERV-K mRNA appears to be mediated by a cis-acting signal on the mRNA, the RcRE, and the protein Rec-these are analogous to the RRE-Rev system in HIV-1. Interestingly, the HIV-1 Rev protein is able to mediate the nuclear export of the HERV-K RcRE, contributing to elevated HERV-K expression in HIV-infected patients. We aimed to understand the structural basis for HIV Rev-HERV-K RcRE recognition. We examined the conformation of the RcRE RNA in solution using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). We found that the 433-nt long RcRE can assume folded or extended conformations as observed by AFM. SAXS analysis of a truncated RcRE variant revealed an "A"-shaped topological structure similar to the one previously reported for the HIV-1 RRE. The effect of the overall topology was examined using several deletion variants. SAXS and biochemical analyses demonstrated that the "A" shape is necessary for efficient Rev-RcRE complex formation in vitro and nuclear export activity in cell culture. The findings provide insight into the mechanism of HERV-K expression and a structural explanation for HIV-1 Rev-mediated expression of HERV-K in HIV-infected patients. IMPORTANCE: Expression of the human endogenous retrovirus type K (HERV-K) has been associated with various cancers and autoimmune diseases. Nuclear export of both HIV-1 and HERV-K mRNAs is dependent on the interaction between a small viral protein (Rev in HIV-1 and Rec in HERV-K) and a region on the mRNA (RRE in HIV-1 and RcRE in HERV-K). HIV-1 Rev is able to mediate the nuclear export of RcRE-containing HERV-K mRNAs, which contributes to elevated production of HERV-K proteins in HIV-infected patients. We report the solution conformation of the RcRE RNA-the first three-dimensional topological structure for a HERV molecule-and find that the RcRE resembles the HIV-1 nuclear export signal, RRE. The finding reveals the structural basis for the increased HERV-K expression observed in HIV-infected patients. Elevated HERV expression, mediated by HIV infection or other stressors, can have various HERV-related biological consequences. The findings provide structural insight for regulation of HERV-K expression.
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http://dx.doi.org/10.1016/j.jmb.2020.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842262PMC
December 2020

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal.

J Biol Chem 2020 10 13;295(42):14391-14401. Epub 2020 Aug 13.

Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, USA

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native MS to investigate RNA binding of wild-type (WT) Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.
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http://dx.doi.org/10.1074/jbc.RA120.014835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573273PMC
October 2020

The heart of the HIV RNA packaging signal?

Authors:
Alan Rein

Proc Natl Acad Sci U S A 2020 08 28;117(33):19621-19623. Epub 2020 Jul 28.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702

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http://dx.doi.org/10.1073/pnas.2013378117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443930PMC
August 2020

Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA.

Viruses 2020 04 2;12(4). Epub 2020 Apr 2.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag-RNA and Gag-Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.
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http://dx.doi.org/10.3390/v12040394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232488PMC
April 2020

IFITM3 Reduces Retroviral Envelope Abundance and Function and Is Counteracted by glycoGag.

mBio 2020 01 21;11(1). Epub 2020 Jan 21.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA

Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3. The viral envelope glycoprotein, known as "Env" in , is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.
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http://dx.doi.org/10.1128/mBio.03088-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974572PMC
January 2020

Nucleic acid-induced dimerization of HIV-1 Gag protein.

J Biol Chem 2019 11 30;294(45):16480-16493. Epub 2019 Sep 30.

Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. , it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.
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http://dx.doi.org/10.1074/jbc.RA119.010580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851336PMC
November 2019

RNA Packaging in HIV.

Authors:
Alan Rein

Trends Microbiol 2019 08 10;27(8):715-723. Epub 2019 May 10.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA. Electronic address:

Successful replication of the AIDS retrovirus, HIV, requires that its genomic RNA be packaged in assembling virus particles with high fidelity. However, cellular mRNAs can also be packaged under some conditions. Viral RNA (vRNA) contains a 'packaging signal' (ψ) and is packaged as a dimer, with two vRNA monomers joined by a limited number of base pairs. It has two conformers, only one of which is capable of dimerization and packaging. Recent years have seen important progress on the 3D structure of dimeric ψ. Gag, the protein that assembles into the virus particle, interacts specifically with ψ, but this is obscured under physiological conditions by its high nonspecific affinity for any RNA. New results suggest that vRNA is selected for packaging because ψ nucleates assembly more efficiently than other RNAs.
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http://dx.doi.org/10.1016/j.tim.2019.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625830PMC
August 2019

Structure and architecture of immature and mature murine leukemia virus capsids.

Proc Natl Acad Sci U S A 2018 12 26;115(50):E11751-E11760. Epub 2018 Nov 26.

Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany;

Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.
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http://dx.doi.org/10.1073/pnas.1811580115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294937PMC
December 2018

Efficient support of virus-like particle assembly by the HIV-1 packaging signal.

Elife 2018 08 2;7. Epub 2018 Aug 2.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, United States.

The principal structural component of a retrovirus particle is the Gag protein. Retroviral genomic RNAs contain a 'packaging signal' ('Ψ') and are packaged in virus particles with very high selectivity. However, if no genomic RNA is present, Gag assembles into particles containing cellular mRNA molecules. The mechanism by which genomic RNA is normally selected during virus assembly is not understood. We previously reported (Comas-Garcia et al., 2017) that at physiological ionic strength, recombinant HIV-1 Gag binds with similar affinities to RNAs with or without Ψ, and proposed that genomic RNA is selectively packaged because binding to Ψ initiates particle assembly more efficiently than other RNAs. We now present data directly supporting this hypothesis. We also show that one or more short stretches of unpaired G residues are important elements of Ψ; Ψ may not be localized to a single structural element, but is probably distributed over >100 bases.
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http://dx.doi.org/10.7554/eLife.38438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092119PMC
August 2018

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element.

J Virol 2017 Nov 13;91(21). Epub 2017 Oct 13.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA

The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other. Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A" shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.
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http://dx.doi.org/10.1128/JVI.00746-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640835PMC
November 2017

Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

Elife 2017 07 20;6. Epub 2017 Jul 20.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, United States.

Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.
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http://dx.doi.org/10.7554/eLife.27055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531834PMC
July 2017

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

J Virol 2017 08 27;91(16). Epub 2017 Jul 27.

Viral Recombination Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.
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http://dx.doi.org/10.1128/JVI.02319-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533913PMC
August 2017

Inhibitors of STAT3, β-catenin, and IGF-1R sensitize mouse PIK3CA-mutant breast cancer to PI3K inhibitors.

Mol Oncol 2017 05 6;11(5):552-566. Epub 2017 Apr 6.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Although mutations in the phosphoinositide 3-kinase catalytic subunit (PIK3CA) are common in breast cancer, PI3K inhibitors alone have shown modest efficacy. We sought to identify additional pathways altered in PIK3CA-mutant tumors that might be targeted in combination with PI3K inhibitors. We generated two transgenic mouse models expressing the human PIK3CA-H1047R- and the -E545K hotspot-mutant genes in the mammary gland and evaluated their effects on development and tumor formation. Molecular analysis identified pathways altered in these mutant tumors, which were also targeted in multiple cell lines derived from the PIK3CA tumors. Finally, public databases were analyzed to determine whether novel pathways identified in the mouse tumors were altered in human tumors harboring mutant PIK3CA. Mutant mice showed increased branching and delayed involution of the mammary gland compared to parental FVB/N mice. Mammary tumors arose in 30% of the MMTV-PIK3CA-H1047R and in 13% of -E545K mice. Compared to MMTV-Her-2 transgenic mouse mammary tumors, H1047R tumors showed increased upregulation of Wnt/β-catenin/Axin2, hepatocyte growth factor (Hgf)/Stat3, insulin-like growth factor 2 (Igf-2), and Igf-1R pathways. Inhibitors of STAT3, β-catenin, and IGF-1R sensitized H1047R-derived mouse tumor cells and PIK3CA-H1047R overexpressing human HS578T breast cancer cells to the cytotoxic effects of PI3K inhibitors. Analysis of The Cancer Genome Atlas database showed that, unlike primary PIK3CA-wild-type and HER-2 breast carcinomas, PIK3CA-mutant tumors display increased expression of AXIN2, HGF, STAT3, IGF-1, and IGF-2 mRNA and activation of AKT, IGF1-MTOR, and WNT canonical signaling pathways. Drugs targeting additional pathways that are altered in PIK3CA-mutant tumors may improve treatment regimens using PI3K inhibitors alone.
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http://dx.doi.org/10.1002/1878-0261.12053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527464PMC
May 2017

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

mBio 2016 11 22;7(6). Epub 2016 Nov 22.

HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, USA

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called "glycogag" (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.

Importance: Many murine leukemia viruses (MLVs) encode a protein called "glycogag." The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5.
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http://dx.doi.org/10.1128/mBio.01985-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120145PMC
November 2016

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus.

Small 2016 Nov 16;12(42):5862-5872. Epub 2016 Sep 16.

Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN, 47405, USA.

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.
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http://dx.doi.org/10.1002/smll.201601712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6810630PMC
November 2016

On the Selective Packaging of Genomic RNA by HIV-1.

Viruses 2016 09 12;8(9). Epub 2016 Sep 12.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.

Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag-gRNA complex will nucleate particle assembly more efficiently than other Gag-RNA complexes. New data shows that among cellular mRNAs, those with long 3'-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3'-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.
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http://dx.doi.org/10.3390/v8090246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035960PMC
September 2016

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation.

J Virol 2016 May 14;90(9):4544-4555. Epub 2016 Apr 14.

Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA

Unlabelled: By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals--electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities.

Importance: Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.
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http://dx.doi.org/10.1128/JVI.02820-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836311PMC
May 2016

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

J Virol 2016 02 4;90(4):1773-87. Epub 2015 Dec 4.

HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, Maryland, USA

Unlabelled: HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization.

Importance: Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.
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http://dx.doi.org/10.1128/JVI.02061-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733982PMC
February 2016

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein.

J Virol 2015 Oct 5;89(20):10371-82. Epub 2015 Aug 5.

Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA.

Unlabelled: Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

Importance: Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.
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http://dx.doi.org/10.1128/JVI.01628-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580166PMC
October 2015

Assay for Cytidine Deaminase Activity of APOBEC3 Protein.

Bio Protoc 2014 Oct;4(20)

Center for Cancer Research, National Cancer Institute, Frederick, USA.

Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA diting enzyme, atalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are an important element in cellular defenses against retrovirus replication, and this "restriction" of retroviral infections is partially due to the cytidine deaminase activity of the APOBEC3. The present protocol (Nair , 2014) describes the assay to detect the deaminase activity of mouse APOBEC3 protein, which targets cytidines present in TCC or TTC motifs in a single-stranded DNA substrate. In brief, the protein preparation to be assayed is incubated with a fluorophore-labeled oligodeoxynucleotide containing the deamination target motif (radiolabeled oligonucleotide substrates have also been successfully used by other groups). Cytidines in the oligonucleotide are deaminated to uridines; the addition of Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, generating an abasic (AB) site in the oligonucleotide. Mild alkali treatment cleaves the substrate oligonucleotide at the AB site; cleaved products are resolved from uncleaved substrate by denaturing polyacrylamide gel electrophoresis and visualized on a fluorescence scanner. The protocol described here is mainly adapted from that described by Iwatani (2006) with modifications. The assay can, of course, be used to detect the activity of other APOBEC3 deaminases targeting DNA substrates, using oligonucleotides containing the cytidine-containing target sequence for the deaminase.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972333PMC
http://dx.doi.org/10.21769/bioprotoc.1266DOI Listing
October 2014

The Notch pathway inhibits TGFβ signaling in breast cancer through HEYL-mediated crosstalk.

Cancer Res 2014 Nov 12;74(22):6509-18. Epub 2014 Sep 12.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233182PMC
November 2014

Antiretroviral restriction factors in mice.

Virus Res 2014 Nov 10;193:130-4. Epub 2014 Jul 10.

HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. Electronic address:

One of the most exciting areas in contemporary retrovirus research is the discovery of "restriction factors". These are cellular proteins that act after virus entry to inhibit infection by or replication of retroviruses (and other viruses and intracellular pathogens). We briefly discuss here three antiretroviral restriction factors in mice: Fv1, APOBEC3, and tetherin, touching on both biological and molecular aspects of these restriction systems.
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http://dx.doi.org/10.1016/j.virusres.2014.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254267PMC
November 2014

An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

J Virol 2014 Jun 12;88(11):6061-8. Epub 2014 Mar 12.

Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA.

Unlabelled: Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles.

Importance: The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.
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http://dx.doi.org/10.1128/JVI.03277-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093898PMC
June 2014

Biochemical and biological studies of mouse APOBEC3.

J Virol 2014 Apr 22;88(7):3850-60. Epub 2014 Jan 22.

HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.

Unlabelled: Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (ΔVif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase-mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3' ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ΔVif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5' end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation.

Importance: One way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells.
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http://dx.doi.org/10.1128/JVI.03456-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993542PMC
April 2014

A conformational transition observed in single HIV-1 Gag molecules during in vitro assembly of virus-like particles.

J Virol 2014 Mar 8;88(6):3577-85. Epub 2014 Jan 8.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA.

Unlabelled: The conformational changes within single HIV-1 Gag molecules that occur during assembly into immature viruses are poorly understood. Using an in vitro assembly assay, it has been proposed that HIV-1 Gag undergoes a conformational transition from a compact conformation in solution to an extended rod-like conformation in virus-like particles (VLPs). Here we used single-molecule Förster resonance energy transfer (smFRET) to test this model by directly probing the conformation of single HIV-1 Gag molecules. We demonstrate that monomeric HIV-1 Gag lacking the p6 domain and the N-terminal myristoyl moiety is found in solution predominantly in a compact conformation. Gag in this conformation, and in the presence of nucleic acid, assembles into 30-nm-diameter particles. However, with the addition of inositol hexakisphosphate, Gag adopts a linear conformation and assembles into full-sized ∼100-to-150-nm-diameter VLPs. Parallel fluorescence correlation spectroscopy measurements show that this conformational transition occurs early in the assembly process when Gag oligomers are small, perhaps as early as upon dimerization. Thus, smFRET measurements confirm that HIV-1 Gag transitions from a compact to a linear conformation during the formation of VLPs. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid.

Importance: The establishment of single-molecule fluorescence techniques reveals the conformational state of individual HIV-1 Gag molecules prior to and during in vitro assembly into virus-like particles. The data demonstrate that Gag in distinct conformations forms particles with different morphologies. In the compact conformation, in the presence of nucleic acid, Gag forms spherical particles of a diameter of approximately 30 nm. In the extended conformation, Gag forms spherical virus-like particles of approximately 100-nm diameter. The adoption of the extended conformation required the presence of inositol hexakisphosphate in addition to nucleic acid. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid.
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http://dx.doi.org/10.1128/JVI.03353-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957938PMC
March 2014

Differential inhibitory effects of cyanovirin-N, griffithsin, and scytovirin on entry mediated by envelopes of gammaretroviruses and deltaretroviruses.

J Virol 2014 Feb 27;88(4):2327-32. Epub 2013 Nov 27.

Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.

The antiviral lectins griffithsin (GRFT), cyanovirin-N (CV-N), and scytovirin (SVN), which inhibit several enveloped viruses, including lentiviruses, were examined for their ability to inhibit entry mediated by Env proteins of delta- and gammaretroviruses. The glycoproteins from human T-cell leukemia virus type 1 (HTLV-1) were resistant to the antiviral effects of all three lectins. For gammaretroviruses, CV-N inhibited entry mediated by some but not all of the envelopes examined, whereas GRFT and SVN displayed only little or no effect.
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http://dx.doi.org/10.1128/JVI.02553-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911537PMC
February 2014

An unusual topological structure of the HIV-1 Rev response element.

Cell 2013 Oct 24;155(3):594-605. Epub 2013 Oct 24.

Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.
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http://dx.doi.org/10.1016/j.cell.2013.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918456PMC
October 2013

Murine leukemia virus p12 functions include hitchhiking into the nucleus.

Authors:
Alan Rein

Proc Natl Acad Sci U S A 2013 Jun 24;110(23):9195-6. Epub 2013 May 24.

HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.

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http://dx.doi.org/10.1073/pnas.1307399110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677489PMC
June 2013

Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex.

Proc Natl Acad Sci U S A 2013 May 13;110(22):9078-83. Epub 2013 May 13.

Department of Microbiology, Abramson Cancer Center and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19103, USA.

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.
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http://dx.doi.org/10.1073/pnas.1217399110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670389PMC
May 2013