Michael F. Summers

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Name: Summers, Michael
Organization: University of Maryland Baltimore County , USA
Department: Howard Hughes Medical Institute and Department of Chemistry and Biochemistry
Title: Professor(PhD)
Co-reporter:Sarah C. Keane;Sayo McCowin;Verna Van;Justin Santos;Heather M. Frank;Xiao Heng;Carly A. Sciandra
PNAS 2016 Volume 113 (Issue 46 ) pp:13033-13038
Publication Date(Web):2016-11-15
DOI:10.1073/pnas.1614785113
HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.
Co-reporter:Siarhei Kharytonchyk;Sarah Monti;Philip J. Smaldino;Joshua D. Brown;Verna Van;Canessa Swanson;Alex Shuey;Alice Telesnitsky;Nicholas C. Bolden;Emily Russo
PNAS 2016 Volume 113 (Issue 47 ) pp:13378-13383
Publication Date(Web):2016-11-22
DOI:10.1073/pnas.1616627113
The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3–R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine (Cap1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines (Cap2G or Cap3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer–dimer equilibrium properties of Cap1G, Cap2G, and Cap3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap1G 5′-leader RNA adopts a dimeric structure, the Cap2G and Cap3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimer-promoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine.
Co-reporter:Sarah C. Keane;Xiao Heng;Siarhei Kharytonchyk;Kun Lu;Venkateswaran Ramakrishnan;Gregory Carter;Shawn Barton;Alyssa Florwick;Azra Hosic;Justin Santos;Nicholas C. Bolden;Sayo McCowin;David A. Case;Bruce A. Johnson;Marco Salemi;Alice Telesnitsky
Science 2015 Volume 348(Issue 6237) pp:917-921
Publication Date(Web):22 May 2015
DOI:10.1126/science.aaa9266

Structural signals that direct HIV packaging

During the viral replication cycle of HIV, unspliced dimeric RNA genomes are efficiently packaged into new virions at the host cell membrane. Packaging is directed by a region at the start of the genome, the 5′ leader. The architecture of the 5′ leader remains controversial. Keane et al. developed nuclear magnetic resonance methods to determine the structure of a 155-nucleotide-long region of the 5′ leader that can direct viral packaging. The structure shows how the 5′ leader binds to the HIV protein that directs packaging, how unspliced dimeric genomes are selected for packaging, and how translation is suppressed when the genome dimerizes.

Science, this issue p. 917

Co-reporter:Kun Lu;Xiao Heng;Lianko Garyu;Sarah Monti;Eric L. Garcia;Siarhei Kharytonchyk;Bilguujin Dorjsuren;Gowry Kulandaivel;Simonne Jones;Atheeth Hiremath;Sai Sachin Divakaruni;Courtney LaCotti;Shawn Barton;Daniel Tummillo;Azra Hosic;Kedy Edme;Sara Albrecht;Alice Telesnitsky
Science 2011 Vol 334(6053) pp:242-245
Publication Date(Web):14 Oct 2011
DOI:10.1126/science.1210460

An RNA structural switch regulates whether the HIV genome is translated or dimerized and packaged.

Co-reporter:Shardell Spriggs, Lianko Garyu, Ryan Connor and Michael F. Summers
Biochemistry 2008 Volume 47(Issue 49) pp:13064-13073
Publication Date(Web):November 12, 2008
DOI:10.1021/bi8014373
The 5′-untranslated region (5′-UTR) of the human immunodeficiency virus type-1 (HIV-1) genome regulates multiple RNA-dependent functions during viral replication and has been proposed to adopt multiple secondary structures. Recent phylogenetic studies identified base pair complementarity between residues of the unique 5′ element and those near the gag start codon (gagAUG) that is conserved among evolutionarily distant retroviruses, suggesting a potential long-range RNA−RNA interaction. However, nucleotide accessibility studies led to conflicting conclusions about the presence of such interactions in virions and in infected cells. Here, we show that an 11-nucleotide oligo-RNA spanning residues 105−115 of the 5′-UTR (U5) readily binds to oligoribonucleotides containing the gag start codon (AUG), disrupting a pre-existing stem loop and forming a heteroduplex stabilized by 11 Watson−Crick base pairs (Kd = 0.47 ± 0.16 μM). Addition of the HIV-1 nucleocapsid protein (NC), the trans-acting viral factor required for genome packaging, disrupts the heteroduplex by binding tightly to U5 (Kd = 122 ± 10 nM). The structure of the NC:U5 complex, determined by NMR, exhibits features similar to those observed in NC complexes with HIV-1 stem loop RNAs, including the insertion of guanosine nucleobases to hydrophobic clefts on the surface of the zinc fingers and a 3′-to-5′ orientation of the RNA relative to protein. Our findings indicate that the previously proposed long-range U5-gagAUG interaction is feasible and suggest a potential NC-dependent mechanism for modulating the structure of the 5′-UTR.
Co-reporter:Michael F. Summers;Freeman A. Hrabowski III
Science 2006 Vol 311(5769) pp:1870-1871
Publication Date(Web):31 Mar 2006
DOI:10.1126/science.1125257

Summary

An undergraduate program involving mentorship, summer and other workshops, and targeting high-achieving high school students improves participation of underrepresented minorities in science.

Co-reporter:Victoria D'Souza & Michael F. Summers
Nature Reviews Microbiology 2005 3(8) pp:643
Publication Date(Web):
DOI:10.1038/nrmicro1210
As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA genomes are selected for packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of genome packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein–RNA complexes are helping to unravel the mechanism of genome packaging and providing the first glimpses of the initial stages of retrovirus assembly.
Co-reporter:Victoria D'Souza & Michael F. Summers
Nature 2004 431(7008) pp:586
Publication Date(Web):
DOI:10.1038/nature02944
Co-reporter:Erin Loeliger;Chun Tang;Paz Luncsford;Dorothy Beckett;Isaac Kinde
PNAS 2004 Volume 101 (Issue 2 ) pp:517-522
Publication Date(Web):2004-01-13
DOI:10.1073/pnas.0305665101
The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (K assoc = 2.5 ± 0.6 × 108 M–2). The equilibrium constant is shifted ≈20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.
Co-reporter:
Nature Structural and Molecular Biology 2002 9(7) pp:537 - 543
Publication Date(Web):28 May 2002
DOI:10.1038/nsb806
Co-reporter:Roberto N. De Guzman;Ryan B. Turner
Biopolymers 1998 Volume 48(Issue 2-3) pp:
Publication Date(Web):26 OCT 2000
DOI:10.1002/(SICI)1097-0282(1998)48:2<181::AID-BIP7>3.0.CO;2-L

The x-ray structure of the glutamine aminoacyl tRNA synthetase bound to its cognate tRNAGln and ATP was reported by Steitz and co-workers in 1989, providing the first high resolution structure of a protein–RNA complex. Since then, high resolution structures have been reported for RNA complexes with five other tRNA synthetases, the elongation factor Tu, the bacteriophage MS2 coat protein, the human spliceosomal U1A and U2B″–U1A′ proteins, and the HIV-1 nucleocapsid protein. Although the number of high resolution structures of protein–RNA complexes are rather small, some general themes have begun to emerge regarding the nature and mechanisms of protein–RNA recognition. © 1999 John Wiley & Sons, Inc. Biopoly 48: 181–195, 1998

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Nature Structural and Molecular Biology 1998 5(7) pp:551 - 555
Publication Date(Web):
DOI:10.1038/805
Co-reporter:Sampson K. Kyere, Peter Y. Mercredi, Xinhong Dong, Paul Spearman, Michael F. Summers
Virus Research (November 2012) Volume 169(Issue 2) pp:411-414
Publication Date(Web):1 November 2012
DOI:10.1016/j.virusres.2012.06.007
During the late phase of the Human Immunodeficiency Virus Type-1 (HIV-1) replication cycle, viral Gag proteins and the intact RNA genome are trafficked to specific sub-cellular membranes where virus assembly and budding occurs. Targeting to the plasma membranes of T cells and macrophages is mediated by interactions between the N-terminal matrix (MA) domain of Gag and cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] molecules. However, in macrophages and dendritic cells, a subset of Gag proteins appears to be targeted to tetraspanin enriched viral compartments, a process that appears to be mediated by MA interactions with the Delta subunit of the cellular Adaptor Protein AP-3 (AP-3δ). We cloned, overexpressed and purified the protein interactive domain of AP-3δ and probed for MA binding by NMR. Unexpectedly, no evidence of binding was observed in these in vitro experiments, even at relatively high protein concentrations (200 μM), suggesting that AP-3δ plays an alternative role in HIV-1 assembly.Highlights► Previously proposed AP-3:MA binding model tested by NMR. ► Findings unexpectedly show that the protein interactive domain of AP-3 does not bind HIV-1 MA. ► Findings suggest AP-3 may play other roles in Gag trafficking and HIV-1 assembly.
Co-reporter:F. Zehra Yildiz, Kathlene Babalola, Michael F. Summers
Virus Research (February 2013) Volume 171(Issue 2) pp:278-286
Publication Date(Web):1 February 2013
DOI:10.1016/j.virusres.2012.07.020
Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of bovine leukemia virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369–U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism.Highlights► Previously proposed packaging signals tested. ► Only one of several proposed packaging elements binds NC with high-affinity. ► NC binding element unexpectedly lacks features common to other retroviral NC binding sites.
Co-reporter:Xiao Heng, Siarhei Kharytonchyk, Eric L. Garcia, Kun Lu, ... Michael F. Summers
Journal of Molecular Biology (30 March 2012) Volume 417(Issue 3) pp:224-239
Publication Date(Web):30 March 2012
DOI:10.1016/j.jmb.2012.01.033
Assembly of human immunodeficiency virus type 1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5′-leader (5′-L) of the viral RNA. Recent studies revealed that the native 5′-L exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5′-L that are important for packaging, we generated HIV-1 5′-L RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding, and packaging. Our findings identify a 159-residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5′-L and contains elements required for efficient RNA packaging.Download high-res image (78KB)Download full-size imageHighlights► A 159-nucleotide region of the HIV-1 5′-L that exhibits NMR spectral features and dimerization and NC binding properties similar to those of the intact leader is identified. ► The 159-nucleotide “core encapsidation signal” is required for efficient and selective packaging of heterologous RNAs into virus particles. ► Packaging defects observed here and elsewhere upon deletion of the TAR hairpin are due to unexpected misfolding of the monomeric form of the 5′-L.
Co-reporter:Jamil S. Saad, Sherimay D. Ablan, Ruba H. Ghanam, Andrew Kim, ... Michael F. Summers
Journal of Molecular Biology (3 October 2008) Volume 382(Issue 2) pp:434-447
Publication Date(Web):3 October 2008
DOI:10.1016/j.jmb.2008.07.027
During the late phase of retroviral replication, newly synthesized Gag proteins are targeted to the plasma membrane (PM), where they assemble and bud to form immature virus particles. Membrane targeting by human immunodeficiency virus type 1 (HIV-1) Gag is mediated by the PM marker molecule phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], which is capable of binding to the matrix (MA) domain of Gag in an extended lipid conformation and of triggering myristate exposure. Here, we show that, as observed previously for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel formed by side chains of helices 1, 2, 3, and 5. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration-dependent exposure. Soluble PI(4,5)P2 analogs containing truncated acyl chains bind HIV-2 MA and induce minor long-range structural changes but do not trigger myristate exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P2 localization. Our findings indicate that HIV-1 and HIV-2 are both targeted to the PM for assembly via a PI(4,5)P2-dependent mechanism, despite differences in the sensitivity of the MA myristyl switch, and suggest a potential mechanism that may contribute to the poor replication kinetics of HIV-2.
Co-reporter:Peter Y. Mercredi, Nadine Bucca, Burk Loeliger, Christy R. Gaines, ... Michael F. Summers
Journal of Molecular Biology (24 April 2016) Volume 428(Issue 8) pp:1637-1655
Publication Date(Web):24 April 2016
DOI:10.1016/j.jmb.2016.03.005
•The influences of lipid constituents on membrane binding by HIV-1 MA were determined.•NMR results differed from those obtained by non-equilibrium flotation assays.•The structural basis for PI(4,5)P2-dependent membrane targeting was re-examined.•Native PI(4,5)P2 does not bind MA in a predicted “extended lipid” conformation.•MA may bind non-raft regions and recruit raft-like constituents during assembly.Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane, where they co-localize and bud to form immature particles. Membrane targeting is mediated by the N-terminally myristoylated matrix (MA) domain of Gag and is dependent on the plasma membrane marker phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Recent studies revealed that PI(4,5)P2 molecules containing truncated acyl chains [tr-PI(4,5)P2] are capable of binding MA in an “extended lipid” conformation and promoting myristoyl exposure. Here we report that tr-PI(4,5)P2 molecules also readily bind to non-membrane proteins, including HIV-1 capsid, which prompted us to re-examine MA–PI(4,5)P2 interactions using native lipids and membrane mimetic liposomes and bicelles. Liposome binding trends observed using a recently developed NMR approach paralleled results of flotation assays, although the affinities measured under the equilibrium conditions of NMR experiments were significantly higher. Native PI(4,5)P2 enhanced MA binding to liposomes designed to mimic non-raft-like regions of the membrane, suggesting the possibility that binding of the protein to disordered domains may precede Gag association with, or nucleation of, rafts. Studies with bicelles revealed a subset of surface and myr-associated MA residues that are sensitive to native PI(4,5)P2, but cleft residues that interact with the 2′-acyl chains of tr-PI(4,5)P2 molecules in aqueous solution were insensitive to native PI(4,5)P2 in bicelles. Our findings call to question extended-lipid MA:membrane binding models, and instead support a model put forward from coarse-grained simulations indicating that binding is mediated predominantly by dynamic, electrostatic interactions between conserved basic residues of MA and multiple PI(4,5)P2 and phosphatidylserine molecules.Download high-res image (107KB)Download full-size image
Co-reporter:Michael F. Summers, Jonathan Karn
Journal of Molecular Biology (22 July 2011) Volume 410(Issue 4) pp:489-490
Publication Date(Web):22 July 2011
DOI:10.1016/j.jmb.2011.05.001
Co-reporter:Kun Lu, Xiao Heng, Michael F. Summers
Journal of Molecular Biology (22 July 2011) Volume 410(Issue 4) pp:609-633
Publication Date(Web):22 July 2011
DOI:10.1016/j.jmb.2011.04.029
Like all retroviruses, the human immunodeficiency virus selectively packages two copies of its unspliced RNA genome, both of which are utilized for strand-transfer-mediated recombination during reverse transcription—a process that enables rapid evolution under environmental and chemotherapeutic pressures. The viral RNA appears to be selected for packaging as a dimer, and there is evidence that dimerization and packaging are mechanistically coupled. Both processes are mediated by interactions between the nucleocapsid domains of a small number of assembling viral Gag polyproteins and RNA elements within the 5′-untranslated region of the genome. A number of secondary structures have been predicted for regions of the genome that are responsible for packaging, and high-resolution structures have been determined for a few small RNA fragments and protein–RNA complexes. However, major questions regarding the RNA structures (and potentially the structural changes) that are responsible for dimeric genome selection remain unanswered. Here, we review efforts that have been made to identify the molecular determinants and mechanism of human immunodeficiency virus type 1 genome packaging.Download high-res image (67KB)Download full-size image
Co-reporter:Brian N. Kelly, Sampson Kyere, Isaac Kinde, Chun Tang, ... Christopher P. Hill
Journal of Molecular Biology (19 October 2007) Volume 373(Issue 2) pp:355-366
Publication Date(Web):19 October 2007
DOI:10.1016/j.jmb.2007.07.070
The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CAN) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CAN and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.
Co-reporter:Yasuyuki Miyazaki, Rossitza N. Irobalieva, Blanton S. Tolbert, Adjoa Smalls-Mantey, ... Michael F. Summers
Journal of Molecular Biology (17 December 2010) Volume 404(Issue 5) pp:751-772
Publication Date(Web):17 December 2010
DOI:10.1016/j.jmb.2010.09.009
The 5′-untranslated regions of all gammaretroviruses contain a conserved “double-hairpin motif” (ΨCD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming “kissing” interactions stabilized by two intermolecular G–C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([ΨCD]2, 132 nt, 42.8 kDa) using a 2H-edited NMR-spectroscopy-based approach. This approach enabled the detection of 1H–1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H–1H correlated and 1H–13C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D′ and SLC′ to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C′ to SL-D′) that gives rise to an elongated overall shape (ca 95 Å × 45 Å ×  25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [ΨCD]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [ΨCD]2 functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.Download high-res image (138KB)Download full-size imageResearch Highlights► Three-dimensional structure determined for 132 nt RNA involved in retroviral genome packaging. ► Solved by a novel 2H-labeled NMR and cryo-ET approach. ► Simultaneously, the largest NMR structure, smallest cryo-ET structure solved to date. ► Prior models based on chemical probing incompatible with the structure. ► Structure likely functions as a scaffold that exposes protein binding sites.
Co-reporter:Jamil S. Saad, Erin Loeliger, Paz Luncsford, Mellisa Liriano, ... Michael F. Summers
Journal of Molecular Biology (16 February 2007) Volume 366(Issue 2) pp:574-585
Publication Date(Web):16 February 2007
DOI:10.1016/j.jmb.2006.11.068
During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P2 binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P2 is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P2 or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P2 binding. This mutant binds PI(4,5)P2 with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.
Co-reporter:Jing Zhou, Rebecca L. Bean, Volker M. Vogt, Michael Summers
Journal of Molecular Biology (12 January 2007) Volume 365(Issue 2) pp:453-467
Publication Date(Web):12 January 2007
DOI:10.1016/j.jmb.2006.10.013
The 5′-untranslated region (5′-UTR) of retroviral genomes contains elements required for genome packaging during virus assembly. For many retroviruses, the packaging elements reside in non-contiguous segments that span most or all of the 5′-UTR. The Rous sarcoma virus (RSV) is an exception, in that its genome can be packaged efficiently by a relatively short, 82 nt segment of the 5′-UTR called μΨ. The RSV 5′-UTR also contains three translational start codons (AUG-1, AUG-2 and AUG-3) that have been controvertibly implicated in translation initiation and genome packaging, one of which (AUG-3) resides within the μΨ sequence. We demonstrated recently that μΨ is capable of binding to the cognate RSV nucleocapsid protein (NC) with high affinity (dissociation constant Kd ∼ 2 nM), and that residues of AUG-3 are essential for tight binding. We now report the solution structure of the NC:μΨ complex, determined using NMR data obtained for samples containing (13C,15N)-labeled NC and 2H-enriched, nucleotide-specifically protonated RNAs. Upon NC binding, μΨ adopts a stable secondary structure that consists of three stem loops (SL-A, SL-B and SL-C) and an 8 bp stem (O3). Binding is mediated by the two zinc knuckle domains of NC. The N-terminal knuckle interacts with a conserved U(217)GCG tetraloop (a member of the UNCG family; N = A,U,G or C), and the C-terminal zinc knuckle binds to residues that flank SL-A, including residues of AUG-3. Mutations of critical nucleotides in these sequences compromise or abolish viral infectivity. Our studies reveal novel structural features important for NC:RNA binding, and support the hypothesis that AUG-3 is conserved for genome packaging rather than translational control.
Co-reporter:Yasuyuki Miyazaki, Eric L. Garcia, Steven R. King, Kilali Iyalla, ... Michael F. Summers
Journal of Molecular Biology (12 February 2010) Volume 396(Issue 1) pp:141-152
Publication Date(Web):12 February 2010
DOI:10.1016/j.jmb.2009.11.033
Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (ΨWT; residues 147–623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([ΨWT]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (ΨM), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
Cadmium, isotope ofmass 113
Mercury, isotope ofmass 199
Proton
adenylyl-(3'→5')-uridylyl-(3'→5')-guanosine
5-((2S,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione
Uridine 5'-triphosphate
4-Androstenedione
Ethanamine, 2,2'-dithiobis-
Nickelate(4-),[(3aS,4S,8S,9S,14S,16R,19S,20S,21S,22aR,25S)-19-(2-amino-2-oxoethyl)-8,25-bis(carboxymethyl)-2,3,3a,4,8,9,11,12,13,14,16,17,19,20,21,22-hexadecahydro-3a,19-dimethyl-2,11-dioxo-1H-7,10-imino-14,16-methano-21,18:22a,5-dinitrilopyrrolo[2,3-i][1]benzazacyclononadecine-4,9,20-tripropanoato(6-)-kN15,kN23,kN24,kN26]-, hydrogen (1:5), (SP-4-2)-
3-METHYL-6-QUINOXALINAMINE