Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

ABSTRACT Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.

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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.

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Cidofovir Resistance in Vaccinia Virus Is Linked to Diminished Virulencein Mice

Journal of Virology ◽

10.1128/jvi.00605-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9391-9401 ◽

Cited By ~ 48

Author(s):

Graciela Andrei ◽

Don B. Gammon ◽

Pierre Fiten ◽

Erik De Clercq ◽

Ghislain Opdenakker ◽

...

Keyword(s):

Drug Resistance ◽

Vaccinia Virus ◽

Dna Polymerase ◽

In Vitro Selection ◽

Cross Resistance ◽

Gene Encoding ◽

Recombinant Viruses ◽

Substitution Mutations

ABSTRACT Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3′-5′ exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

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Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program

Infection and Immunity ◽

10.1128/iai.00181-07 ◽

2007 ◽

Vol 75 (8) ◽

pp. 4148-4157 ◽

Cited By ~ 57

Author(s):

Oliver Goldmann ◽

Maren von Köckritz-Blickwede ◽

Claudia Höltje ◽

Gursharan S. Chhatwal ◽

Robert Geffers ◽

...

Keyword(s):

Gene Expression ◽

Streptococcus Pyogenes ◽

Interleukin 1 ◽

Alternative Activation ◽

Gene Encoding ◽

Necrosis Factor Alpha ◽

Murine Macrophages ◽

Infected Cells

ABSTRACT The complex response of murine macrophages to infection with Streptococcus pyogenes was investigated at the level of gene expression with a high-density oligomer microarray. More than 400 genes were identified as being differentially regulated. Many of the up-regulated genes encode molecules involved in the immune response and in inflammation, transcription, signaling, apoptosis, the cell cycle, electron transport, and cell adhesion. Of particular interest was the up-regulation of proinflammatory cytokines, typical of the classically activated macrophages (M1 phenotype), such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, and as well as the up-regulation of anti-inflammatory mediators, such as IL-1 decoy receptor and IL-10, associated with alternative macrophage activation (M2 phenotype). Furthermore, the gene encoding inducible nitric oxide synthase (iNOS), an enzyme typically implicated in classical activation, was not induced in infected macrophages. Instead, the gene encoding arginase, a competitor for the iNOS substrate arginine involved in the alternative activation pathway, was up-regulated in S. pyogenes-infected cells. Thus, the microarray-based gene expression analysis demonstrated that S. pyogenes induces an atypical activation program in macrophages, with some but not all features of the classical or alternative activation phenotypes. The microarray data also suggested that the bactericidal activity of macrophages against S. pyogenes is mediated by phagocyte oxidase, as p47phox was up-regulated in infected cells. Indeed, the in vivo and in vitro killing of S. pyogenes was markedly diminished in the absence of functional phagocyte (p47 phox−/−) but not in the absence of iNOS (iNOS−/−). An understanding of how macrophages respond to S. pyogenes at the molecular level may facilitate the development of new therapeutic paradigms.

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Characterization of the Rhesus Cytomegalovirus US28 Locus

Journal of Virology ◽

10.1128/jvi.77.19.10404-10413.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10404-10413 ◽

Cited By ~ 32

Author(s):

M. E. T. Penfold ◽

T. L. Schmidt ◽

D. J. Dairaghi ◽

P. A. Barry ◽

T. J. Schall

Keyword(s):

Viral Entry ◽

Intracellular Signaling ◽

Northern Analysis ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Wide Range ◽

Infected Cells ◽

G Protein Coupled

ABSTRACT Human cytomegalovirus (CMV) US28 (and the related open reading frame [ORF] US27) are G-protein-coupled receptor homologs believed to play a role in viral pathogenesis. In vitro, US28 has been shown to bind and internalize ligands, as well as activate intracellular signaling in response to certain chemokines, and to initiate the migration of smooth muscle cells to chemokine gradients. To assess the role of US28 in vivo, we examined the rhesus model and sequenced and characterized the rhesus CMV US28 locus. We found that rhesus CMV carries five tandem homologs of US28, all widely divergent from US28 and from each other. By reverse transcription-PCR and Northern analysis, we demonstrated expression of these ORFs in infected cells. With stable cell lines expressing these ORFs, we analyzed the homolog's binding and signaling characteristics across a wide range of chemokines and found one (RhUS28.5) to have a ligand binding profile similar to that of US28. In addition, we localized US28 and the rhesus CMV homolog RhUS28.5 to the envelope of infectious virions, suggesting a role in viral entry or cell tropism.

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Vaccinia Virus A34 Glycoprotein Determines the Protein Composition of the Extracellular Virus Envelope

Journal of Virology ◽

10.1128/jvi.01969-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2150-2160 ◽

Cited By ~ 19

Author(s):

Beatriz Perdiguero ◽

María M. Lorenzo ◽

Rafael Blasco

Keyword(s):

Vaccinia Virus ◽

Protein Composition ◽

Recombinant Vaccinia Virus ◽

Virus Envelope ◽

Enveloped Virus ◽

Extracellular Virus ◽

Transmembrane Glycoprotein ◽

C Terminus ◽

Vaccinia Viruses ◽

Infected Cells

ABSTRACT The outer envelope of the extracellular form of vaccinia virus contains five virus-encoded proteins, F13, A33, A34, A56, and B5, that, with the exception of A56, are implicated in virus egress or infectivity. A34, a type II transmembrane glycoprotein, is involved in the induction of actin tails, the release of enveloped virus from the surfaces of infected cells, and the disruption of the virus envelope after ligand binding prior to virus entry. To investigate interactions between A34 and other envelope proteins, a recombinant vaccinia virus (vA34RHA) expressing an epitope-tagged version of A34 (A34HA) was constructed by appending an epitope from influenza virus hemagglutinin to the C terminus of A34. Complexes of A34HA with B5 and A36, but not with A33 or F13, were detected in vA34RHA-infected cells. A series of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A34. Both the cytoplasmic and the transmembrane domains of B5 were dispensable for binding to A34. Most of the extracellular domain of B5, which contains four short consensus repeats homologous to complement control proteins, was sufficient for A34 interaction, indicating that both proteins interact through their ectodomains. Immunofluorescence experiments on cells infected with A34-deficient virus indicated that A34 is required for efficient targeting of B5, A36, and A33 into wrapped virions. Consistent with this observation, the envelope of A34-deficient virus contained normal amounts of F13 but decreased amounts of A33 and B5 with respect to the parental WR virus. These results point to A34 as a major determinant in the protein composition of the vaccinia virus envelope.

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Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement

Journal of Virology ◽

10.1128/jvi.01797-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1201-1215 ◽

Cited By ~ 62

Author(s):

Mohammed Rafii-El-Idrissi Benhnia ◽

Megan M. McCausland ◽

Juan Moyron ◽

John Laudenslager ◽

Steven Granger ◽

...

Keyword(s):

Vaccinia Virus ◽

Protective Mechanism ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Rapid Control ◽

Infected Cells ◽

Sterilizing Immunity ◽

Powerful Mechanism

ABSTRACT Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV). Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection.

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Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein

Journal of General Virology ◽

10.1099/0022-1317-82-11-2747 ◽

2001 ◽

Vol 82 (11) ◽

pp. 2747-2760 ◽

Cited By ~ 81

Author(s):

María M. Geada ◽

Inmaculada Galindo ◽

María M. Lorenzo ◽

Beatriz Perdiguero ◽

Rafael Blasco

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Intracellular Transport ◽

Virus Transport ◽

Enveloped Virus ◽

Vaccinia Viruses ◽

Butanedione Monoxime ◽

Golgi Network ◽

Dependent Mechanism

Vaccinia virus produces several forms of infectious virions. Intracellular mature virions (IMV) assemble in areas close to the cell nucleus. Some IMV acquire an envelope from intracellular membranes derived from the trans-Golgi network, producing enveloped forms found in the cytosol (intracellular enveloped virus; IEV), on the cell surface (cell-associated enveloped virus) or free in the medium (extracellular enveloped virus; EEV). Blockage of IMV envelopment inhibits transport of virions to the cell surface, indicating that enveloped virus forms are required for virion movement from the Golgi area. To date, the induction of actin tails that propel IEV is the only well-characterized mechanism for enveloped virus transport. However, enveloped virus transport and release occur under conditions where actin tails are not formed. In order to study these events, recombinant vaccinia viruses were constructed with GFP fused to the most abundant protein in the EEV envelope, P37 (F13L). The P37–GFP fusion, like normal P37, accumulated in the Golgi area and was incorporated efficiently into enveloped virions. These recombinants allowed the monitoring of enveloped virus movements in vivo. In addition to a variety of relatively slow movements (<0·4 μm/s), faster, saltatory movements both towards and away from the Golgi area were observed. These movements were different from those dependent on actin tails and were inhibited by the microtubule-disrupting drug nocodazole, but not by the myosin inhibitor 2,3-butanedione monoxime. Video microscopy (5 frames per s) revealed that saltatory movements had speeds of up to, and occasionally more than, 3 μm/s. These results suggest that a second, microtubule-dependent mechanism exists for intracellular transport of enveloped vaccinia virions.

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The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model

Journal of General Virology ◽

10.1099/0022-1317-83-11-2833 ◽

2002 ◽

Vol 83 (11) ◽

pp. 2833-2844 ◽

Cited By ~ 54

Author(s):

Julian A. Symons ◽

Elizabeth Adams ◽

David C. Tscharke ◽

Patrick C. Reading ◽

Herman Waldmann ◽

...

Keyword(s):

Vaccinia Virus ◽

Deletion Mutant ◽

Soluble Factor ◽

Gene Encoding ◽

Mouse Splenocytes ◽

Infected Cells ◽

Ifn Γ ◽

Revertant Virus

A bioassay that measured the interleukin (IL)-12-induced production of interferon (IFN)-γ from mouse splenocytes was used to identify a soluble factor in the supernatants of vaccinia virus (VV)-infected cells that inhibited the production of IFN-γ. This soluble factor was expressed by 14 out of 16 VV strains including the Western Reserve (WR) strain, but strains Copenhagen and Tashkent and a mutant of strain WR called 6/2 lacked this activity. The gene encoding this activity was identified as C12L by transferring DNA present in VV WR but missing in VV WR 6/2 into VV Copenhagen and testing for expression of the soluble factor. The C12L protein shows amino acid similarity to IL-18 binding proteins that are encoded by poxviruses, mice and humans, and C12L protein produced from VV or baculovirus inhibited the biological activity of mouse IL-18 in vitro. Thus the inhibition of IL-12-induced IFN-γ production was due to indirect effects of C12L on IL-18, illustrating the synergistic action of these pro-inflammatory cytokines. To study the role of the C12L protein in the virus life-cycle, we constructed a deletion mutant lacking the C12L gene and a revertant virus in which the gene was reinserted into the deletion mutant. In vitro the replication and plaque size of these viruses were indistinguishable. However, infection of BALB/c mice by the intranasal route showed that the deletion mutant was attenuated and induced lower weight loss and signs of illness compared to controls.

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Murine Gammaherpesvirus 68 Open Reading Frame 11 Encodes a Nonessential Virion Component

Journal of Virology ◽

10.1128/jvi.79.5.3163-3168.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 3163-3168 ◽

Cited By ~ 8

Author(s):

Jessica M. Boname ◽

Janet S. May ◽

Philip G. Stevenson

Keyword(s):

Lytic Replication ◽

Open Reading Frame ◽

Reading Frame ◽

Murine Gammaherpesvirus ◽

Infected Cells ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.

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Differential S-acylation of Enveloped Viruses

Protein and Peptide Letters ◽

10.2174/0929866526666190603082521 ◽

2019 ◽

Vol 26 (8) ◽

pp. 588-600

Author(s):

Larisa V. Kordyukova ◽

Marina V. Serebryakova ◽

Vladislav V. Khrustalev ◽

Michael Veit

Keyword(s):

Fatty Acid ◽

Protein Trafficking ◽

Virus Assembly ◽

Covalent Attachment ◽

Post Translational Modification ◽

Enveloped Virus ◽

Infected Cells ◽

Regulate Protein

Post-translational modifications often regulate protein functioning. Covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage (known as protein palmitoylation or S-acylation) affects protein trafficking, protein-protein and protein-membrane interactions. This post-translational modification is coupled to membrane fusion or virus assembly and may affect viral replication in vitro and thus also virus pathogenesis in vivo. In this review we outline modern methods to study S-acylation of viral proteins and to characterize palmitoylproteomes of virus infected cells. The palmitoylation site predictor CSS-palm is critically tested against the Class I enveloped virus proteins. We further focus on identifying the S-acylation sites directly within acyl-peptides and the specific fatty acid (e.g, palmitate, stearate) bound to them using MALDI-TOF MS-based approaches. The fatty acid heterogeneity/ selectivity issue attracts now more attention since the recently published 3D-structures of two DHHC-acyl-transferases gave a hint how this might be achieved.

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