Heat inactivation of vaccinia virus particle-associated functions: properties of heated particles in vivo and in vitro.

AbstractNeutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.

Download Full-text

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Journal of Virology ◽

10.1128/jvi.01593-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12355-12367 ◽

Cited By ~ 32

Author(s):

Mohammed Rafii-El-Idrissi Benhnia ◽

Megan M. McCausland ◽

John Laudenslager ◽

Steven W. Granger ◽

Sandra Rickert ◽

...

Keyword(s):

Antibody Response ◽

Vaccinia Virus ◽

Smallpox Vaccine ◽

Human Antibody ◽

Human Mabs ◽

Complement Components ◽

Vaccinia Virion ◽

Virion Antigen

ABSTRACT Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.

Download Full-text

Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.7.3353-3365.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3353-3365 ◽

Cited By ~ 183

Author(s):

Chi-Long Lin ◽

Che-Sheng Chung ◽

Hans G. Heine ◽

Wen Chang

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

Download Full-text

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433-5441.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

Download Full-text

Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

Journal of Virology ◽

10.1128/jvi.01884-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2540-2552 ◽

Cited By ~ 52

Author(s):

Michael H. Lehmann ◽

Wolfgang Kastenmuller ◽

Judith D. Kandemir ◽

Florian Brandt ◽

Yasemin Suezer ◽

...

Keyword(s):

Vaccinia Virus ◽

Viral Vector ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Human Monocytic Cell Line ◽

Modified Vaccinia Virus Ankara ◽

Ccl2 Expression ◽

Human Monocytic Cell

ABSTRACT Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4+ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.

Download Full-text

By Binding CD80 and CD86, the Vaccinia Virus M2 Protein Blocks Their Interactions with both CD28 and CTLA4 and Potentiates CD80 Binding to PD-L1

Journal of Virology ◽

10.1128/jvi.00207-19 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 4

Author(s):

Patricia Kleinpeter ◽

Christelle Remy-Ziller ◽

Eline Winter ◽

Murielle Gantzer ◽

Virginie Nourtier ◽

...

Keyword(s):

Immune Response ◽

Vaccinia Virus ◽

Cytotoxic T Lymphocyte ◽

Myxoma Virus ◽

Parental Virus ◽

M2 Protein ◽

First Results ◽

Infected Cells

ABSTRACTIn this article we report that the M2 protein encoded by the vaccinia virus is secreted as a homo-oligomer by infected cells and binds two central costimulation molecules, CD80 (B7-1) and CD86 (B7-2). These interactions block the ligation of the two B7 proteins to both soluble CD28 and soluble cytotoxic T-lymphocyte associated protein 4 (CTLA4) but favor the binding of soluble PD-L1 to soluble CD80. M2L gene orthologues are found in several other poxviruses, and the B7-CD28/CTLA4 blocking activity has been identified for several culture supernatants of orthopoxvirus-infected cells and for a recombinant myxoma virus M2 protein homolog (i.e., Gp120-like protein, or Gp120LP). Overall, these data indicate that the M2 poxvirus family of proteins may be involved in immunosuppressive activities broader than the NF-κB inhibition already reported (R. Gedey, X. L. Jin, O. Hinthong, and J. L. Shisler, J Virol 80:8676–8685, 2006, https://doi.org/10.1128/JVI.00935-06). A Copenhagen vaccinia virus with a deletion of the nonessential M2L locus was generated and compared with its parental virus. This M2L-deleted vaccinia virus, unlike the parental virus, does not generate interference with the B7-CD28/CTLA4/PD-L1 interactions. Moreover, this deletion did not affect any key features of the virus (in vitroreplication, oncolytic activitiesin vitroandin vivo,and intratumoral expression of a transgene in an immunocompetent murine model). Altogether, these first results suggest that the M2 protein has the potential to be used as a new immunosuppressive biotherapeutic and that the M2L-deleted vaccinia virus represents an attractive new oncolytic platform with an improved immunological profile.IMPORTANCEThe vaccinia virus harbors in its genome several genes dedicated to the inhibition of the host immune response. Among them, M2L was reported to inhibit the intracellular NF-κB pathway. We report here several new putative immunosuppressive activities of M2 protein. M2 protein is secreted and binds cornerstone costimulatory molecules (CD80/CD86). M2 binding to CD80/CD86 blocks their interaction with soluble CD28/CTLA4 but also favors the soluble PD-L1-CD80 association. These findings open the way for new investigations deciphering the immune system effects of soluble M2 protein. Moreover, a vaccinia virus with a deletion of its M2L has been generated and characterized as a new oncolytic platform. The replication and oncolytic activities of the M2L-deleted vaccinia virus are indistinguishable from those of the parental virus. More investigations are needed to characterize in detail the immune response triggered against both the tumor and the virus by this M2-defective vaccinia virus.

Download Full-text

F11-Mediated Inhibition of RhoA Signalling Enhances the Spread of Vaccinia Virus In Vitro and In Vivo in an Intranasal Mouse Model of Infection

PLoS ONE ◽

10.1371/journal.pone.0008506 ◽

2009 ◽

Vol 4 (12) ◽

pp. e8506 ◽

Cited By ~ 41

Author(s):

João V. Cordeiro ◽

Susana Guerra ◽

Yoshiki Arakawa ◽

Mark P. Dodding ◽

Mariano Esteban ◽

...

Keyword(s):

Mouse Model ◽

Vaccinia Virus ◽

Mouse Model Of Infection

Download Full-text

Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.

The Journal of Cell Biology ◽

10.1083/jcb.132.3.291 ◽

1996 ◽

Vol 132 (3) ◽

pp. 291-298 ◽

Cited By ~ 282

Author(s):

A A McCracken ◽

J L Brodsky

Keyword(s):

Protein Degradation ◽

Atp Hydrolysis ◽

Genetically Engineered ◽

Heat Inactivation ◽

Protein Factor ◽

Transport Vesicles ◽

Alpha Factor ◽

Processed Form

To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radio-labeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of p alpha F was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.

Download Full-text

Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L) replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N)-methanocarbathymidine

Virology Journal ◽

10.1186/1743-422x-5-39 ◽

2008 ◽

Vol 5 (1) ◽

pp. 39 ◽

Cited By ~ 7

Author(s):

Mark N Prichard ◽

Earl R Kern ◽

Debra C Quenelle ◽

Kathy A Keith ◽

Richard W Moyer ◽

...

Keyword(s):

Vaccinia Virus ◽

Antiviral Drug

Download Full-text

Reduced sulphydryl groups are required for DNA binding of Ku protein

Biochemical Journal ◽

10.1042/bj2930769 ◽

1993 ◽

Vol 293 (3) ◽

pp. 769-774 ◽

Cited By ~ 14

Author(s):

W W Zhang ◽

M Yaneva

Keyword(s):

Dna Binding ◽

Protein A ◽

Binding Activity ◽

Heat Inactivation ◽

Cysteine Residues ◽

Dna Binding Activity ◽

Sulphydryl Groups ◽

Ku Protein

The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.

Download Full-text