scholarly journals Across the Hall from Pioneers

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 491
Author(s):  
Alan Rein
Keyword(s):  
Transmembrane Protein ◽  
Zinc Fingers ◽  
Sequence Information ◽  
Particle Assembly ◽  
Nucleocapsid Proteins ◽  
Gag Proteins ◽  
Virus Maturation ◽  
The Us ◽  
Env Protein ◽  
Translational Suppression

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 pol gene expression in gammaretroviruses; amazingly, this is a fundamentally different mechanism of suppression from that in most other retroviral genera.

scholarly journals IgG and IgM antibody formation to spike and nucleocapsid proteins in COVID-19 characterized by multiplex immunoblot assays

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jyotsna Shah ◽  
Song Liu ◽  
Hari-Hara Potula ◽  
Prerna Bhargava ◽  
Iris Cruz ◽  
...  
Keyword(s):  
Viral Infections ◽  
Antibody Responses ◽  
Nucleocapsid Proteins ◽  
Igm Antibodies ◽  
Igm Antibody ◽  
Specificity And Sensitivity ◽  
The Us ◽  
Autoimmune Conditions ◽  
Antibody Positivity ◽  
Recombinant Nucleocapsid Protein

Abstract Background Rapid and simple serological assays for characterizing antibody responses are important in the current COVID-19 pandemic caused by SARS-CoV-2. Multiplex immunoblot (IB) assays termed COVID-19 IB assays were developed for detecting IgG and IgM antibodies to SARS-CoV-2 virus proteins in COVID-19 patients. Methods Recombinant nucleocapsid protein and the S1, S2 and receptor binding domain (RBD) of the spike protein of SARS-CoV-2 were used as target antigens in the COVID-19 IBs. Specificity of the IB assay was established with 231 sera from persons with allergy, unrelated viral infections, autoimmune conditions and suspected tick-borne diseases, and 32 goat antisera to human influenza proteins. IgG and IgM COVID-19 IBs assays were performed on 84 sera obtained at different times after a positive RT-qPCR test from 37 COVID-19 patients with mild symptoms. Results Criteria for determining overall IgG and IgM antibody positivity using the four SARS-CoV-2 proteins were developed by optimizing specificity and sensitivity in the COVID-19 IgG and IgM IB assays. The estimated sensitivities and specificities of the COVID-19 IgG and IgM IBs for IgG and IgM antibodies individually or for either IgG or IgM antibodies meet the US recommendations for laboratory serological diagnostic tests. The proportion of IgM-positive sera from the COVID-19 patients following an RT-qPCR positive test was maximal at 83% before 10 days and decreased to 0% after 100 days, while the proportions of IgG-positive sera tended to plateau between days 11 and 65 at 78–100% and fall to 44% after 100 days. Detection of either IgG or IgM antibodies was better than IgG or IgM alone for assessing seroconversion in COVID-19. Both IgG and IgM antibodies detected RBD less frequently than S1, S2 and N proteins. Conclusions The multiplex COVID-19 IB assays offer many advantages for simultaneously evaluating antibody responses to different SARS-CoV-2 proteins in COVID-19 patients.

scholarly journals Failure to detect evidence of human T-lymphotropic virus (HTLV) type I and type II in blood donors with isolated gag antibodies to HTLV-I/II

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 544-550 ◽  
Author(s):  
RB Lal ◽  
DL Rudolph ◽  
JE Coligan ◽  
SK Brodine ◽  
CR Roberts
Keyword(s):  
Blood Donors ◽  
Armed Forces ◽  
Type I ◽  
Gag Protein ◽  
T Lymphotropic Virus ◽  
The Us ◽  
Polymerase Chain ◽  
Env Protein ◽  
Radioimmunoprecipitation Assay ◽  
Immunodominant Epitopes

Abstract Of the 267,650 blood donations from members of the US armed forces, 72 (0.027%) were serologically confirmed to be positive for human T- lymphotropic virus type I/II (HTLVpos) and 379 (0.14%) were Western blot (WB)-indeterminate with banding pattern restricted to the proteins encoded by the gag gene only (HTLVind). To determine whether these apparently healthy HTLVind blood donors are infected with HTLV-I or HTLV-II, coded specimens from randomly selected military blood donors (n = 73) were tested for antibodies to HTLV by WB and radioimmunoprecipitation assay (RIPA) using HTLV-I (MT-2) antigens, by enzyme immunoassay using synthetic peptides representing the immunodominant epitopes of HTLV, and for sequences of proviral HTLV DNA by the polymerase chain reaction (PCR). Of the 73 HTLVind donors, none showed presence of env reactivity by HTLV WB and RIPA. Minimal reactivity was observed with synthetic immunodominant motifs derived from the env protein of HTLV-I (Env-1(191–214) and Env-5(242–257)) or HTLV-II (Env-2(187–209) and Env-20(85–102)) and gag protein (Gag-1a(102– 117) and Gag-10(364–385)). A peptide corresponding to the endogenous retroviral sequence with structural homologies to the gag protein of HTLVs (RTVLgag) reacted with antibodies not only in HTLVpos (88%) and HTLVind (42% to 66%) specimens, but also reacted with normal control subjects (60%). Furthermore, none of the 73 HTLVind specimens demonstrated presence of the HTLV genome when amplified with primers for the pol and tax/rex region. Six to 23 months from the initial test, 27 subjects still gave indeterminate WB patterns, and 13 of these repeat specimens were still negative for the presence of HTLV genome. We conclude that individuals at low risk for HTLV infection who have HTLVind WB reactivity are rarely, if ever, infected with HTLV-I or HTLV- II.

scholarly journals CryoET structures of immature HIV Gag reveal six-helix bundle

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Luiza Mendonça ◽  
Dapeng Sun ◽  
Jiying Ning ◽  
Jiwei Liu ◽  
Abhay Kotecha ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Single Amino Acid ◽  
Helical Conformation ◽  
Particle Assembly ◽  
Virus Maturation ◽  
Helix Bundle ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Single Amino Acid Mutation

AbstractGag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

scholarly journals Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1140 ◽  
Author(s):  
Ryosuke Matsuura ◽  
Kazunori Inabe ◽  
Hiroyuki Otsuki ◽  
Kazuo Kurokawa ◽  
Naoshi Dohmae ◽  
...  
Keyword(s):  
Life Cycle ◽  
Cell Surface ◽  
Bovine Leukemia Virus ◽  
Transmembrane Protein ◽  
Leukemia Virus ◽  
Viral Life Cycle ◽  
Neoplastic Disease ◽  
Single Mutation ◽  
Early Endosomes ◽  
Env Protein

Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia viruses, is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. The transmembrane subunit of the BLV envelope glycoprotein, gp30, contains three completely conserved YXXL sequences that fit an endocytic sorting motif. The two N-terminal YXXL sequences are reportedly critical for viral infection. However, their actual function in the viral life cycle remains undetermined. Here, we identified the novel roles of each YXXL sequence. Syncytia formation ability was upregulated by a single mutation of the tyrosine (Tyr) residue in any of the three YXXL sequences, indicating that each YXXL sequence is independently able to regulate the fusion event. The alteration resulted from significantly high expression of gp51 on the cell surface, thereby decreasing the amount of gp51 in early endosomes and further revealing that the three YXXL sequences are independently required for internalization of the envelope (Env) protein, following transport to the cell surface. Moreover, the 2nd and 3rd YXXL sequences contributed to Env protein incorporation into the virion by functionally distinct mechanisms. Our findings provide new insights regarding the three YXXL sequences toward the BLV viral life cycle and for developing new anti-BLV drugs.

scholarly journals Molecular cloning and functional expression of bovine spleen ecto-NAD+ glycohydrolase: structural identity with human CD38

1999 ◽  
Vol 345 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Angélique AUGUSTIN ◽  
Hélène MULLER-STEFFNER ◽  
Francis SCHUBER
Keyword(s):  
Transmembrane Protein ◽  
Functional Expression ◽  
Full Length ◽  
Cysteine Residues ◽  
Sequence Information ◽  
Functional Domain ◽  
High Sequence Identity ◽  
Full Length Cdna ◽  
Nad Glycohydrolase ◽  
C Terminus

Bovine spleen ecto-NAD+ glycohydrolase, an archetypal member of the mammalian membrane-associated NAD(P)+ glycohydrolase enzyme family (EC 3.2.2.6), displays catalytic features similar to those of CD38, i.e. a protein originally described as a lymphocyte differentiation marker involved in the metabolism of cyclic ADP-ribose and signal transduction. Using amino acid sequence information obtained from NAD+ glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a full-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD+ glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular and contains two potential N-glycosylation sites, yields the fully catalytically active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD+ glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activities at the surface of the cells. The bovine enzyme, which is the first ‘classical’ NAD(P)+ glycohydrolase whose structure has been established, presents a particularly high sequence identity with CD38, including the presence of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine residues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the catalytic activities of CD38/NAD+ glycohydrolases so far identified. Altogether, our data strongly suggest that the cloned bovine spleen ecto-NAD+ glycohydrolase is the bovine equivalent of CD38.

scholarly journals Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

2007 ◽  
Vol 81 (19) ◽  
pp. 10718-10728 ◽  
Author(s):  
Lisa Z. Scheifele ◽  
Scott P. Kenney ◽  
Tina M. Cairns ◽  
Rebecca C. Craven ◽  
Leslie J. Parent
Keyword(s):  
Virus Particle ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Structural Defects ◽  
High Molecular Weight Fraction ◽  
Particle Assembly ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Virion Core

ABSTRACT Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.

scholarly journals Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7431-7441 ◽  
Author(s):  
Guy Zuber ◽  
Jason McDermott ◽  
Sonya Karanjia ◽  
Weiyi Zhao ◽  
Michael F. Schmid ◽  
...  
Keyword(s):  
Proteolytic Processing ◽  
Nucleocapsid Proteins ◽  
Gag Proteins ◽  
Virus Particles ◽  
His Tag ◽  
Different Types ◽  
Membrane Bound ◽  
Immature Virus ◽  
Local Protein

ABSTRACT Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

scholarly journals Importance of Basic Residues in the Nucleocapsid Sequence for Retrovirus Gag Assembly and Complementation Rescue

1998 ◽  
Vol 72 (11) ◽  
pp. 9034-9044 ◽  
Author(s):  
J. Bradford Bowzard ◽  
Robert P. Bennett ◽  
Neel K. Krishna ◽  
Sandra M. Ernst ◽  
Alan Rein ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Mutational Analysis ◽  
Zinc Fingers ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Gag Proteins ◽  
Amino Terminal ◽  
C Terminus ◽  
Zinc Finger Motifs ◽  
And Function

ABSTRACT The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger motifs and function to provide the proper density to viral particles. There are two zinc fingers and at least two I domains within these Gag proteins. To more thoroughly characterize the important sequence features and properties of I domains, we analyzed Gag proteins that contain one or no zinc finger motifs. Chimeric proteins containing the amino-terminal half of RSV Gag and various portions of the carboxy terminus of murine leukemia virus (MLV) (containing one zinc finger) Gag had only one I domain, whereas similar chimeras with human foamy virus (HFV) (containing no zinc fingers) Gag had at least two. Mutational analysis of the MLV NC sequence and inspection of I domain sequences within the zinc-fingerless C terminus of HFV Gag suggested that clusters of basic residues, but not the zinc finger motif residues themselves, are required for the formation of particles of proper density. In support of this, a simple string of strongly basic residues was found to be able to substitute for the RSV I domains. We also explored the possibility that differences in I domains (e.g., their number) account for differences in the ability of Gag proteins to be rescued into particles when they are unable to bind to membranes. Previously published experiments have shown that such membrane-binding mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with RSV-MLV chimeras now map this difference to the NC sequence of MLV. Importantly, the same RSV-MLV chimeras could be rescued by complementation when the block to budding was after, rather than before, transport to the membrane. These results suggest that MLV Gag molecules begin to interact at a much later time after synthesis than those of RSV and HIV.

scholarly journals CryoET structures of immature HIV Gag reveal a complete six-helix bundle and stabilizing small molecules distinct from IP6

2020 ◽  
Author(s):  
Luiza Mendonça ◽  
Dapeng Sun ◽  
Jiying Ning ◽  
Jiwei Liu ◽  
Abhay Kotecha ◽  
...  
Keyword(s):  
Small Molecules ◽  
Structural Basis ◽  
Particle Assembly ◽  
Virus Maturation ◽  
Helix Bundle ◽  
Polyprotein Precursor ◽  
Subtomogram Averaging ◽  
Gag Assembly ◽  
Hiv 1

AbstractGag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.

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