scholarly journals The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Lentiviral Bipartite Nuclear Localization Signal Harboring an Atypical Spacer Sequence

2009 ◽  
Vol 83 (24) ◽  
pp. 12842-12853 ◽  
Author(s):  
Andrea Gomez Corredor ◽  
Denis Archambault
Keyword(s):  
Protein Identification ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
Bovine Immunodeficiency Virus ◽  
Immunodeficiency Virus ◽  
Arginine Residues ◽  
Localization Signal ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Rev Protein

ABSTRACT The bovine immunodeficiency virus (BIV) Rev protein (186 amino acids [aa] in length) is involved in the nuclear exportation of partially spliced and unspliced viral RNAs. Previous studies have shown that BIV Rev localizes in the nucleus and nucleolus of infected cells. Here we report the characterization of the nuclear/nucleolar localization signals (NLS/NoLS) of this protein. Through transfection of a series of deletion mutants of BIV Rev fused to enhanced green fluorescent protein and fluorescence microscopy analyses, we were able to map the NLS region between aa 71 and 110 of the protein. Remarkably, by conducting alanine substitution of basic residues within the aa 71 to 110 sequence, we demonstrated that the BIV Rev NLS is bipartite, maps to aa 71 to 74 and 95 to 101, and is predominantly composed of arginine residues. This is the first report of a bipartite Rev (or Rev-like) NLS in a lentivirus/retrovirus. Moreover, this NLS is atypical, as the length of the sequence between the motifs composing the bipartite NLS, e.g., the spacer sequence, is 20 aa. Further mutagenesis experiments also identified the NoLS region of BIV Rev. It localizes mainly within the NLS spacer sequence. In addition, the BIV Rev NoLS sequence differs from the consensus sequence reported for other viral and cellular nucleolar proteins. In summary, we conclude that the nucleolar and nuclear localizations of BIV Rev are mediated via novel NLS and NoLS motifs.

scholarly journals Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 900
Author(s):  
Marlène Labrecque ◽  
Claude Marchand ◽  
Denis Archambault
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Encephalitis Virus ◽  
Viral Mrnas ◽  
Caprine Arthritis Encephalitis Virus ◽  
Cell Compartments ◽  
Arginine Residues ◽  
Green Fluorescent ◽  
Rev Protein

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein. These signals are key actors in the nucleocytoplasmic shuttling of a lentiviral Rev protein. Several deletion and alanine substitution mutants were generated from a plasmid encoding the CAEV Rev wild-type protein that was fused to the enhanced green fluorescent protein (EGFP). Following cell transfection, images were captured by confocal microscopy and the fluorescence was quantified in the different cell compartments. The results showed that the NLS region is localized between amino acids (aa) 59 to 75, has a monopartite-like structure and is exclusively composed of arginine residues. The NoLS was found to be partially associated with the NLS. Finally, the CAEV Rev protein’s NES mapped between aa 89 to 101, with an aa spacing between the hydrophobic residues that was found to be unconventional as compared to that of other retroviral Rev/Rev-like proteins.

scholarly journals Bidirectional Increase in Permeability of Nuclear Envelope upon Poliovirus Infection and Accompanying Alterations of Nuclear Pores

2004 ◽  
Vol 78 (18) ◽  
pp. 10166-10177 ◽  
Author(s):  
George A. Belov ◽  
Peter V. Lidsky ◽  
Olga V. Mikitas ◽  
Denise Egger ◽  
Konstantin A. Lukyanov ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Protein ◽  
Fluorescent Protein ◽  
Cyclin B1 ◽  
Nuclear Proteins ◽  
Nuclear Pores ◽  
Poliovirus Infection ◽  
Localization Signal ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3×EGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-l-alanyl-l-alanyl-l-prolyl-l-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.

scholarly journals Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes

2002 ◽  
Vol 277 (51) ◽  
pp. 49562-49568 ◽  
Author(s):  
Katsuhiko Nakashima ◽  
Teruki Hagiwara ◽  
Michiyuki Yamada
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Calcium Ionophore ◽  
Subcellular Fractionation ◽  
Peptidylarginine Deiminase ◽  
Sequence Feature ◽  
Link Protein ◽  
Arginine Residues ◽  
Localization Signal ◽  
Green Fluorescent

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca2+dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45–74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophoreA23187, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.

Recombinant Simian Immunodeficiency Virus Expressing Green Fluorescent Protein Identifies Infected Cells in Rhesus Monkeys

1999 ◽  
Vol 15 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Louis Alexander ◽  
Ronald S. Veazey ◽  
Susan Czajak ◽  
Maryann Demaria ◽  
Michael Rosenzweig ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Simian Immunodeficiency Virus ◽  
Rhesus Monkeys ◽  
Fluorescent Protein ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Green Fluorescent

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus

2008 ◽  
Vol 89 (3) ◽  
pp. 676-686 ◽  
Author(s):  
Kristi Tamm ◽  
Andres Merits ◽  
Inga Sarand
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Structural Protein ◽  
Arginine Residues ◽  
Localization Signal ◽  
Infected Cells ◽  
Replicase Complex

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

Confocal Analysis of the Distribution and Persistence of Sindbis Virus (TaV-GFP) Infection in Midguts of Aedes aegypti Mosquitoes

2020 ◽  
Vol 26 (2) ◽  
pp. 267-274
Author(s):  
Jason J. Saredy ◽  
Florence Y. Chim ◽  
Zoë L. Lyski ◽  
Yani P. Ahearn ◽  
Doria F. Bowers
Keyword(s):  
Aedes Aegypti ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Blood Feeding ◽  
Target Tissue ◽  
Secondary Target ◽  
Posterior Midgut ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Global Threat

AbstractBiological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

scholarly journals Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Prerna Vohra ◽  
Christina Vrettou ◽  
Jayne C. Hope ◽  
John Hopkins ◽  
Mark P. Stevens
Keyword(s):  
Lymph Nodes ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Large Animal ◽  
Ileal Mucosa ◽  
Zoonotic Infections ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Salmonella Enterica Serovar Dublin

AbstractSalmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

scholarly journals High Physiological Levels of LMP1 Result in Phosphorylation of eIF2α in Epstein-Barr Virus-Infected Cells

2004 ◽  
Vol 78 (4) ◽  
pp. 1657-1664 ◽  
Author(s):  
Ngan Lam ◽  
Mark L. Sandberg ◽  
Bill Sugden
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Barr Virus ◽  
Signaling Domain ◽  
Infected Cells ◽  
Epstein Barr ◽  
Green Fluorescent ◽  
Carboxy Terminal

ABSTRACT LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J. 17:1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol. 74:9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene 19:1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2α in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2α was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2α. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.

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