scholarly journals The Equine Herpesvirus 1 US2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

1999 ◽  
Vol 73 (4) ◽  
pp. 3430-3437 ◽  
Author(s):  
Alexandra Meindl ◽  
Nikolaus Osterrieder
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Viral Envelope ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Amino Terminal ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 US2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 US2 protein specifically detected a protein with an M r of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-M r Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-M r Us2 polypeptide. Irrespective of its size, the US2 protein was incorporated into virions. The EHV-1 US2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 US2 protein or to a truncated US2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 US2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the US2 protein in the viral envelope and plasma membrane of infected cells, a US2-negative RacL11 mutant (L11ΔUS2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a US2-repaired virus. After infection of BALB/c mice with L11ΔUS2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 US2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

scholarly journals Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Panda ◽  
Debasis Nayak ◽  
Asit K. Pattnaik
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Viral Envelope ◽  
M Protein ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

scholarly journals Peptide transport activity of the transporter associated with antigen processing (TAP) is inhibited by an early protein of equine herpesvirus-1

2004 ◽  
Vol 85 (2) ◽  
pp. 349-353 ◽  
Author(s):  
Aruna P. N. Ambagala ◽  
Raju S. Gopinath ◽  
S. Srikumaran
Keyword(s):  
Antigen Processing ◽  
Protein S ◽  
Class I ◽  
Peptide Transport ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Early Protein ◽  
Infected Cells ◽  
Herpesvirus 1

Equine herpesvirus-1 (EHV-1) downregulates surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. The objective of this study was to investigate whether EHV-1 interferes with peptide translocation by the transporter associated with antigen processing (TAP) and to identify the proteins responsible. Using an in vitro transport assay, we showed that EHV-1 inhibited transport of peptides by TAP as early as 2 h post-infection (p.i). Complete shutdown of peptide transport was observed by 8 h p.i. Furthermore, pulse–chase experiments revealed that maturation of class I molecules in the endoplasmic reticulum (ER) was delayed in EHV-1-infected cells, which may be due to reduced availability of peptides in the ER as a result of TAP inhibition. Metabolic inhibition studies indicated that an early protein(s) of EHV-1 is responsible for this effect.

scholarly journals Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts

2001 ◽  
Vol 82 (8) ◽  
pp. 1951-1957 ◽  
Author(s):  
Karen M. van der Meulen ◽  
Hans J. Nauwynck ◽  
Maurice B. Pensaert
Keyword(s):  
Cell Cycle ◽  
Mononuclear Cells ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Mitogen Stimulation ◽  
Blood Mononuclear Cells ◽  
Intercellular Contacts ◽  
M Phase ◽  
Infected Cells ◽  
Herpesvirus 1

In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38·4±4·5% were CD5+ T-lymphocytes (18·1±3·2% CD4+ 13·6±1·8% CD8+), 18·1±5·4% were B-lymphocytes, 8·5±3·9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0·974) and G2/M-phase (r=0·927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G2/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1·6 to 13·4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.

scholarly journals An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

1996 ◽  
Vol 132 (5) ◽  
pp. 795-811 ◽  
Author(s):  
M M Sauter ◽  
A Pelchen-Matthews ◽  
R Bron ◽  
M Marsh ◽  
C C LaBranche ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Simian Immunodeficiency Virus ◽  
Cytoplasmic Domain ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Coated Pits ◽  
Immunodeficiency Virus ◽  
Infected Cells

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

scholarly journals The α-TIF (VP16) Homologue (ETIF) of Equine Herpesvirus 1 Is Essential for Secondary Envelopment and Virus Egress

2006 ◽  
Vol 80 (6) ◽  
pp. 2609-2620 ◽  
Author(s):  
Jens von Einem ◽  
Daniel Schumacher ◽  
Dennis J. O'Callaghan ◽  
Nikolaus Osterrieder
Keyword(s):  
Cell Line ◽  
Infectious Virus ◽  
Early Gene ◽  
Growth Defect ◽  
Main Role ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Ultrastructural Studies ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT The equine herpesvirus 1 (EHV-1) α-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11ΔETIF) was constructed and analyzed. After transfection of vL11ΔETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11ΔETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11ΔETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11ΔETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11ΔETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.

scholarly journals Equine Herpesvirus 1 Entry via Endocytosis Is Facilitated by αV Integrins and an RSD Motif in Glycoprotein D

2008 ◽  
Vol 82 (23) ◽  
pp. 11859-11868 ◽  
Author(s):  
Gerlinde R. Van de Walle ◽  
Sarah T. Peters ◽  
Brian C. VanderVen ◽  
Dennis J. O'Callaghan ◽  
Nikolaus Osterrieder
Keyword(s):  
Plasma Membrane ◽  
Mononuclear Cells ◽  
Mutational Analysis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Endocytic Pathway ◽  
Equine Herpesvirus ◽  
Glycoprotein D ◽  
Equine Herpesvirus 1 ◽  
Herpesvirus 1

ABSTRACT Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as αVβ5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.

scholarly journals The Gene 10 (UL49.5) Product of Equine Herpesvirus 1 Is Necessary and Sufficient for Functional Processing of Glycoprotein M

2002 ◽  
Vol 76 (6) ◽  
pp. 2952-2963 ◽  
Author(s):  
Jens Rudolph ◽  
Christian Seyboldt ◽  
Harald Granzow ◽  
Nikolaus Osterrieder
Keyword(s):  
Homologous Gene ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Mature Form ◽  
Growth Defects ◽  
Plaque Phenotype ◽  
Infected Cells ◽  
Herpesvirus 1 ◽  
Necessary And Sufficient ◽  
Golgi Network

ABSTRACT The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-β-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyAΔ49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyAΔ49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyAΔ49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M r protein was coprecipitated with gM in KyA- or KyAΔ49.5R-infected cells or virions. This protein was absent in cells infected with KyΔ49.5 or KyAΔgM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.

scholarly journals Expression of the Full-Length Form of gp2 of Equine Herpesvirus 1 (EHV-1) Completely Restores Respiratory Virulence to the Attenuated EHV-1 Strain KyA in CBA Mice

2005 ◽  
Vol 79 (8) ◽  
pp. 5105-5115 ◽  
Author(s):  
Patrick M. Smith ◽  
Shannon M. Kahan ◽  
Colin B. Rorex ◽  
Jens von Einem ◽  
Nikolaus Osterrieder ◽  
...  
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
T Cell Activation ◽  
Full Length ◽  
Equine Herpesvirus ◽  
Wild Type ◽  
Rnase Protection ◽  
Equine Herpesvirus 1 ◽  
Cba Mice ◽  
Herpesvirus 1

ABSTRACT Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, interferon γ-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.

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