Pig BVDV-2 non-structural protein (Npro) links to cellular antiviral response in vitro

Human immunodeficiency virus type 1 (HIV-1) has evolved various measures to counter the host cell's innate antiviral response during the course of infection. Interferon (IFN)-stimulated gene products are produced following HIV-1 infection to limit viral replication, but viral proteins and RNAs counteract their effect. One such mechanism is specifically directed against the IFN-induced Protein Kinase PKR, which is centrally important to the cellular antiviral response. In the presence of viral RNAs, PKR is activated and phosphorylates the translation initiation factor eIF2α. This shuts down the synthesis of both host and viral proteins, allowing the cell to mount an effective antiviral response. PACT (protein activator of PKR) is a cellular protein activator of PKR, primarily functioning to activate PKR in response to cellular stress. Recent studies have indicated that during HIV-1 infection, PACT's normal cellular function is compromised and that PACT is unable to activate PKR. Using various reporter systems and in vitro kinase assays, we establish in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. Our results highlight an important pathway by which HIV-1 transcripts subvert the host cell's antiviral activities to enhance their translation.

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Regulation of a double-stranded RNA modification activity in human cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3719 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3719-3725 ◽

Cited By ~ 9

Author(s):

L M Morrissey ◽

K Kirkegaard

Keyword(s):

Antiviral Response ◽

Human Cells ◽

Rna Modification ◽

Double Stranded Rna ◽

Modification Activity ◽

Antiviral Function ◽

Cellular Antiviral Response ◽

In Cells

A double-stranded RNA (dsRNA)-specific modification activity from Xenopus oocytes and human cells dsRNA modifier) converts adenosine residues present in dsRNA to inosines. The function of the dsRNA modifier is unknown, although it has been suggested that it may be part of the cellular antiviral response. We investigated the relationship between the activity of the dsRNA modifier, viral infection, and the antiviral response in human cells induced by poly(rI)-poly(rC) [poly(I.C)] treatment. We found, unexpectedly, that treatment of HeLa cells with poly(I.C) or other dsRNA molecules resulted in the dramatic inhibition of the dsRNA modifier. Mixing experiments, reconstruction experiments, and pretreatment of extracts with RNases indicated that inhibition of the dsRNA modifier did not result from the continued presence of a soluble inhibitor such as dsRNA) in the in vitro modification reactions. Treatment of cells with cyclohexamide or dactinomycin simultaneously with the poly(I.C) demonstrated that in vivo inhibition of the dsRNA modifier did not require new transcription or translation. The dsRNA modification activity was also substantially inhibited in cells infected with poliovirus and was slightly inhibited in cells infected with adenovirus. The inhibition of the dsRNA modifier during the antiviral state is thus not consistent with an antiviral function, and instead suggests another cellular function for dsRNA modification.

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Regulation of a double-stranded RNA modification activity in human cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3719-3725.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3719-3725

Author(s):

L M Morrissey ◽

K Kirkegaard

Keyword(s):

Antiviral Response ◽

Human Cells ◽

Rna Modification ◽

Double Stranded Rna ◽

Modification Activity ◽

Antiviral Function ◽

Cellular Antiviral Response ◽

In Cells

A double-stranded RNA (dsRNA)-specific modification activity from Xenopus oocytes and human cells dsRNA modifier) converts adenosine residues present in dsRNA to inosines. The function of the dsRNA modifier is unknown, although it has been suggested that it may be part of the cellular antiviral response. We investigated the relationship between the activity of the dsRNA modifier, viral infection, and the antiviral response in human cells induced by poly(rI)-poly(rC) [poly(I.C)] treatment. We found, unexpectedly, that treatment of HeLa cells with poly(I.C) or other dsRNA molecules resulted in the dramatic inhibition of the dsRNA modifier. Mixing experiments, reconstruction experiments, and pretreatment of extracts with RNases indicated that inhibition of the dsRNA modifier did not result from the continued presence of a soluble inhibitor such as dsRNA) in the in vitro modification reactions. Treatment of cells with cyclohexamide or dactinomycin simultaneously with the poly(I.C) demonstrated that in vivo inhibition of the dsRNA modifier did not require new transcription or translation. The dsRNA modification activity was also substantially inhibited in cells infected with poliovirus and was slightly inhibited in cells infected with adenovirus. The inhibition of the dsRNA modifier during the antiviral state is thus not consistent with an antiviral function, and instead suggests another cellular function for dsRNA modification.

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Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

Journal of Bacteriology ◽

10.1128/jb.183.4.1423-1433.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 28

Author(s):

Susan R. Heimer ◽

Harry L. T. Mobley

Keyword(s):

Proteus Mirabilis ◽

Accessory Proteins ◽

Structural Protein ◽

Yeast Two Hybrid ◽

Nickel Ions ◽

Catalytically Active ◽

Tract Infections ◽

Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

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Df31 is a novel nuclear protein involved in chromatin structure in Drosophila melanogaster

Journal of Cell Science ◽

10.1242/jcs.114.1.37 ◽

2001 ◽

Vol 114 (1) ◽

pp. 37-47 ◽

Cited By ~ 1

Author(s):

G. Crevel ◽

H. Huikeshoven ◽

S. Cotterill

Keyword(s):

Cell Types ◽

Drosophila Embryo ◽

Cell Fractionation ◽

General Function ◽

Structural Protein ◽

Chromosomal Structure ◽

Binding Characteristics ◽

Wide Range ◽

Cellular Dna

We originally isolated the Df31 protein from Drosophila embryo extracts as a factor which could decondense Xenopus sperm, by removing the sperm specific proteins and interacting with histones to facilitate their loading onto DNA. We now believe that this protein has a more general function in cellular DNA metabolism. The Df31 gene encodes a very hydrophilic protein with a predicted molecular mass of 18.5 kDa. Immunostaining showed that Df31 was present in a wide range of cell types throughout differentiation and in both dividing and non-dividing cells. In all cases the protein is present in large amounts, comparable with the level of nucleosomes. Injection of antisense oligonucleotides to lower the level of Df31 in embryos caused severe disruption of the nuclear structure. Large irregular clumps of DNA were formed, and in most cases the amount of DNA associated with each clump was more than that found in a normal nucleus. Immunofluorescence, cell fractionation, and formaldehyde cross-linking show that Df31 is associated with chromatin and that a significant fraction of it binds very tightly. It also shows the same binding characteristics when loaded onto chromatin in vitro. Chromatin fractionation shows that Df31 is tightly associated with nucleosomes, preferentially with oligonucleosomes. Despite this no differences were observed in the properties of nucleosomes loaded in the in vitro system in the presence and absence of Df31. These results suggest that Df31 has a role in chromosomal structure, most likely acting as a structural protein at levels of folding higher than that of nucleosomes.

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Caspofungin and LTX-315 inhibit SARS-CoV-2 replication by targeting the nsp12 polymerase

10.21203/rs.3.rs-19872/v1 ◽

2020 ◽

Author(s):

Min Wang ◽

Fei Ye ◽

Jiaqi Su ◽

Jingru Zhao ◽

Bin Yuan ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Vero Cells ◽

Polymerase Activity ◽

Structural Protein ◽

Live Virus ◽

Drug Candidates ◽

Virtual Screen ◽

Virus Assay ◽

Promising Target

Abstract The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously designated as 2019-nCoV) outbreak has caused global concern1. Currently, there are no clinically approved specific drugs or vaccines available for this virus. The viral polymerase is a promising target for developing broad- spectrum antiviral drugs. Here, based on the highly similar structure of SARS- CoV non-structural protein 12 (nsp12) polymerase subunit2, we applied virtual screen for the available compounds, including both the FDA-approved and under- clinic drugs, to identify potential antiviral molecules against SARS-CoV-2. We found two drugs, the clinically approved anti-fungi drug Caspofungin Acetate (Cancidas) and the oncolytic peptide LTX-315, can bind SARS-CoV-2 nsp12 protein to block the polymerase activity in vitro. Further live virus assay revealed that both Caspofungin Acetate and LTX-315 can effectively inhibit SARS-CoV-2 replication in vero cells. These findings present promising drug candidates for treatment of related diseases and would also stimulate the development of pan- coronavirus antiviral agents.Authors Min Wang, Fei Ye, Jiaqi Su, Jingru Zhao, and Bin Yuan contributed equally to this work.

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The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.79.21.13463-13472.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13463-13472 ◽

Cited By ~ 69

Author(s):

Danso Ako-Adjei ◽

Marc C. Johnson ◽

Volker M. Vogt

Keyword(s):

Nuclear Export ◽

Leukemia Virus ◽

Particle Diameter ◽

Particle Morphology ◽

Structural Protein ◽

Wild Type ◽

Virus Like Particles ◽

Gag Proteins ◽

Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

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Expression in non-lens tissues of an enzyme activity related to the ‘lens-specific’ protein, delta crystallin

Development ◽

10.1242/dev.111.1.181 ◽

1991 ◽

Vol 111 (1) ◽

pp. 181-190

Author(s):

D.I. de Pomerai ◽

W.K. Ip ◽

M. McLaughlin ◽

K.C. Perry

Keyword(s):

Ectopic Expression ◽

Molecular Size ◽

Protein Band ◽

Major Constituent ◽

Lens Protein ◽

Related Protein ◽

Structural Protein ◽

Cycle Enzyme ◽

Published Evidence

When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, delta crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick delta 1 crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick delta crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the delta-related protein band. So-called ‘ectopic’ expression of delta crystallin in embryonic NR (and other tissues) may thus involve the delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.

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