scholarly journals The Vaccinia Virus E8R Gene Product: a Viral Membrane Protein That Is Made Early in Infection and Packaged into the Virions' Core

2002 ◽  
Vol 76 (19) ◽  
pp. 9773-9786 ◽  
Author(s):  
Laura Doglio ◽  
Ario De Marco ◽  
Sibylle Schleich ◽  
Norbert Roos ◽  
Jacomine Krijnse Locker
Keyword(s):  
Life Cycle ◽  
Membrane Protein ◽  
Vaccinia Virus ◽  
Gene Product ◽  
Viral Entry ◽  
Hela Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Dna Viruses ◽  
Replication Sites

ABSTRACT Vaccinia virus (VV), a member of the poxvirus family, is unique among most other DNA viruses in that both transcription and DNA replication occur in the cytoplasm of the host cell. It was recently shown by electron microscopy (EM) that soon after viral DNA synthesis is initiated in HeLa cells, the replication sites become enwrapped by the membrane of the endoplasmic reticulum (ER). In the same study, a novel VV membrane protein, the E8R gene product, that may play a role in the ER wrapping process was identified (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001). In the present study, the gene product of E8R was characterized both biochemically and morphologically. We show that E8R is made predominantly early in infection but is packaged into the virion. On two-dimensional gel electrophoresis, the protein appeared as a single spot throughout the VV life cycle; however, in the assembled virion, the protein underwent several modifications which resulted in a change in its molecular weight and its isoelectric point. EM of labeled cryosections of infected HeLa cells showed that the protein localized to the ER and to membranes located on one side of the Golgi complex as early as 1 h postinfection. Late in infection, E8R was additionally associated with membranes of immature virions and with intracellular mature viruses. Although E8R is predominantly associated with membranes, we show that the protein is associated with viral cores; the protein is present in cores made with NP-40-dithiothreitol as well as in incoming cores, the result of the viral entry process, early in infection. Finally, we show that E8R can be phosphorylated in vitro by the viral kinase F10L. It is able to bind DNA in vitro, and this binding may be modulated by phosphorylation by F10L. A putative role of the E8R gene product throughout the VV life cycle is discussed.

scholarly journals Quantitative SUMO-1 Modification of a Vaccinia Virus Protein Is Required for Its Specific Localization and Prevents Its Self-Association

2005 ◽  
Vol 16 (6) ◽  
pp. 2822-2835 ◽  
Author(s):  
Silvia Palacios ◽  
Laurent H. Perez ◽  
Sonja Welsch ◽  
Sibylle Schleich ◽  
Katarzyna Chmielarska ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Gene Product ◽  
Virus Protein ◽  
Dna Viruses ◽  
Cytosolic Side ◽  
Specific Localization ◽  
Replication Sites ◽  
Self Association

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1–conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral “mini-nuclei” and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.

scholarly journals Characterization of Specific Immune Responses of Mice Inoculated with Recombinant Vaccinia Virus Expressing an 18-Kilodalton Outer Membrane Protein of Brucella abortus

2000 ◽  
Vol 7 (1) ◽  
pp. 114-118 ◽  
Author(s):  
Ramesh Vemulapalli ◽  
Silvio Cravero ◽  
Christine L. Calvert ◽  
Thomas E. Toth ◽  
Nammalwar Sriranganathan ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Brucella Abortus ◽  
Virulent Strain ◽  
Shuttle Vector ◽  
Protein Fusion

ABSTRACT Using the shuttle vector pMCO2 and the vaccinia virus wild-type WR strain, we constructed a recombinant virus expressing an 18-kDa outer membrane protein of Brucella abortus. BALB/c mice inoculated with this virus produced 18-kDa protein-specific antibodies, mostly of immunoglobulin G2a isotype, and in vitro stimulation of splenocytes from these mice with purified maltose binding protein–18-kDa protein fusion resulted in lymphocyte proliferation and gamma interferon production. However, these mice were not protected against a challenge with the virulent strain B. abortus2308. Disruption of the 18-kDa protein's gene in vaccine strainB. abortus RB51 did not affect either the strain's protective capabilities or its in vivo attenuation characteristics. These observations suggest that the 18-kDa protein plays no role in protective immunity.

scholarly journals Ebolavirus Entry: From Molecular Characterization to Drug Discovery

2019 ◽  
Author(s):  
Cristiano Salata ◽  
Arianna Calistri ◽  
Gualtiero Alvisi ◽  
Michele Celestino ◽  
Cristina Parolin ◽  
...  
Keyword(s):  
Life Cycle ◽  
Viral Entry ◽  
Large Scale ◽  
Ebola Virus ◽  
Virus Disease ◽  
Drug Repurposing ◽  
In Vitro Study ◽  
Rational Drug Design ◽  
Global Public Health

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and caused by members of the Filoviridae family. The recent large outbreak of EVD in West Africa (2013-2016), highlighted the worldwide danger of this disease and its impact on global public health and economy. The development of highly needed anti-Filoviridae antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, and therefore screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to in vitro study of Filoviridae entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus like particles, tremendously boosted both our knowledge on viral life cycle and the identification of promising anti-Filoviridae compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.

scholarly journals Role of Viral Factor E3L in Modified Vaccinia Virus Ankara Infection of Human HeLa Cells: Regulation of the Virus Life Cycle and Identification of Differentially Expressed Host Genes

2005 ◽  
Vol 79 (4) ◽  
pp. 2584-2596 ◽  
Author(s):  
Holger Ludwig ◽  
Jörg Mages ◽  
Caroline Staib ◽  
Michael H. Lehmann ◽  
Roland Lang ◽  
...  
Keyword(s):  
Life Cycle ◽  
Vaccinia Virus ◽  
Hela Cells ◽  
Immune Defense ◽  
Oligoadenylate Synthetase ◽  
Molecular Monitoring ◽  
Late Gene Expression ◽  
Modified Vaccinia Virus Ankara ◽  
Inducible Protein

ABSTRACT Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-ΔE3L, was found defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2′-5′-oligoadenylate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-ΔE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-ΔE3L infection. Interestingly, a cluster of genes up regulated exclusively in MVA-ΔE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible protein β, and dual-specificity protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle.

scholarly journals A Novel Cellular Protein, VPEF, Facilitates Vaccinia Virus Penetration into HeLa Cells through Fluid Phase Endocytosis

2008 ◽  
Vol 82 (16) ◽  
pp. 7988-7999 ◽  
Author(s):  
Cheng-Yen Huang ◽  
Tsai-Yi Lu ◽  
Chi-Horng Bair ◽  
Yuan-Shau Chang ◽  
Jeng-Kuan Jwo ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Hela Cells ◽  
Intracellular Transport ◽  
Fluid Phase ◽  
Cellular Protein ◽  
Cell Entry ◽  
Surface Lipid ◽  
Fluid Phase Endocytosis

ABSTRACT Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.

scholarly journals Phytochemicals: Potential Lead Compounds for COVID-19 Therapeutics

2021 ◽  
Vol 15 ◽  
Author(s):  
Srishti Kashyap ◽  
Revathy Nadhan ◽  
Danny N. Dhanasekaran
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Viral Infection ◽  
Viral Entry ◽  
Fatal Outcome ◽  
Epigallocatechin Gallate ◽  
Host Cells ◽  
Host Responses ◽  
Global Pandemic

Coronavirus Disease 2019 (COVID-19) is a global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2). The rising number of cases of this highly transmissible infection has pressed for the urgent need to find effective therapeutics. The life cycle of SARS-CoV-2 includes the viral entry, viral replication, viral assembly and release. The symptoms associated with viral infection often leads to fatal outcome with pneumonia, myocarditis, acute respiratory distress syndrome, hypercoagulability, and/or multi-organ failure. Recent studies have reported that phytochemicals such as emodin, epigallocatechin gallate, and berberine could, albeit modestly, inhibit different stages of SARS-CoV-2 life cycle. The phytochemicals have been shown to disrupt viral infection and replication by blocking viral-surface spike protein binding to entry receptor angiotensin-converting enzyme (ACE2), inhibiting viral membrane fusion with host cells, inhibiting RNA-dependent RNA polymerase involved in viral replication, and/or pathological host- responses in vitro. The focus of this review is to evaluate the efficacies of these phytochemicals on inhibiting SARS-CoV-2 viral infection, growth, or disease progression as well as to provide a perspective on the potential use of these phytochemicals in the development of novel therapeutics against SARS-CoV-2

scholarly journals The Block in Assembly of Modified Vaccinia Virus Ankara in HeLa Cells Reveals New Insights into Vaccinia Virus Morphogenesis

2002 ◽  
Vol 76 (16) ◽  
pp. 8318-8334 ◽  
Author(s):  
M. Carmen Sancho ◽  
Sibylle Schleich ◽  
Gareth Griffiths ◽  
Jacomine Krijnse-Locker
Keyword(s):  
Vaccinia Virus ◽  
Hela Cells ◽  
Immunofluorescence Microscopy ◽  
Close Association ◽  
Viral Dna ◽  
Virus Morphogenesis ◽  
Modified Vaccinia Virus Ankara ◽  
Replication Sites ◽  
Mature Virus ◽  
H 30

ABSTRACT It has previously been shown that upon infection of HeLa cells with modified vaccinia virus Ankara (MVA), assembly is blocked at a late stage of infection and immature virions (IVs) accumulate (G. Sutter and B. Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992). In the present study the morphogenesis of MVA in HeLa cells was studied in more detail and compared to that under two conditions that permit the production of infectious particles: infection of HeLa cells with the WR strain of vaccinia virus (VV) and infection of BHK cells with MVA. Using several quantitative and qualitative assays, we show that early in infection, MVA in HeLa cells behaves in a manner identical to that under the permissive conditions. By immunofluorescence microscopy (IF) at late times of infection, the labelings for an abundant membrane protein of the intracellular mature virus, p16/A14L, and the viral DNA colocalize under permissive conditions, whereas in HeLa cells infected with MVA these two structures do not colocalize to the same extent. In both permissive and nonpermissive infection, p16-labeled IVs first appear at 5 h postinfection. In HeLa cells infected with MVA, IVs accumulated predominantly outside the DNA regions, whereas under permissive conditions they were associated with the viral DNA. At 4 h 30 min, the earliest time at which p16 is detected, the p16 labeling was found predominantly in a small number of distinct puncta by IF, which were distinct from the sites of DNA in both permissive and nonpermissive infection. By electron microscopy, no crescents or IVs were found at this time, and the p16-labeled structures were found to consist of membrane-rich vesicles that were in continuity with the cellular endoplasmic reticulum. Over the next 30 min of infection, a large number of p16-labeled crescents and IVs appeared abruptly under both permissive and nonpermissive conditions. Under permissive conditions, these IVs were in close association with the sites of DNA, and a significant amount of these IVs engulfed the viral DNA. In contrast, under nonpermissive conditions, the IVs and DNA were mostly in separate locations and relatively few IVs acquired DNA. Our data show that in HeLa cells MVA forms normal DNA replication sites and normal viral precursor membranes but the transport between these two structures is inhibited.

scholarly journals Ebola Virus Entry: From Molecular Characterization to Drug Discovery

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 274 ◽  
Author(s):  
Cristiano Salata ◽  
Arianna Calistri ◽  
Gualtiero Alvisi ◽  
Michele Celestino ◽  
Cristina Parolin ◽  
...  
Keyword(s):  
Life Cycle ◽  
Viral Entry ◽  
Large Scale ◽  
Ebola Virus ◽  
Virus Disease ◽  
Virus Entry ◽  
Drug Repurposing ◽  
Rational Drug Design ◽  
Global Public Health

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013–2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.

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