scholarly journals Vaccinia Virus 15-Kilodalton (A14L) Protein Is Essential for Assembly and Attachment of Viral Crescents to Virosomes

1998 ◽  
Vol 72 (2) ◽  
pp. 1287-1296 ◽  
Author(s):  
Juan Ramón Rodríguez ◽  
Cristina Risco ◽  
José L. Carrascosa ◽  
Mariano Esteban ◽  
Dolores Rodríguez
Keyword(s):  
Vaccinia Virus ◽  
Electron Microscopic Examination ◽  
Viral Proteins ◽  
Wild Type ◽  
Electron Microscopic ◽  
Small Plaque ◽  
Virion Assembly ◽  
Plaque Phenotype ◽  
Infected Cells ◽  
Intermediate Compartment

ABSTRACT Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacIoperator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-β-d-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interespersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

scholarly journals Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.

1991 ◽  
Vol 174 (6) ◽  
pp. 1629-1637 ◽  
Author(s):  
J H Cox ◽  
J R Bennink ◽  
J W Yewdell
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Antigen Presentation ◽  
Viral Proteins ◽  
Genetically Engineered ◽  
Class I ◽  
Wild Type ◽  
Histocompatibility Complex ◽  
Infected Cells ◽  
Lumenal Domain

The E3/19K glycoprotein of adenovirus functions to diminish recognition of adenovirus-infected cells by major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) by binding intracellular class I molecules and preventing them from reaching the plasma membrane. In the present study we have characterized the nature of the interaction between E3/19K and the H-2Kd (Kd) molecule. An E3/19K molecule genetically engineered to terminate six residues from its normal COOH terminus (delta E19), was found to associate with Kd in a manner indistinguishable from wild-type E3/19K. Unlike E3/19K, however, delta E19 was transported through the Golgi complex to the plasma membrane, where it could be detected biochemically and immunocytochemically using a monoclonal antibody specific for the lumenal domain of E3/19K. Importantly, delta E19 also differed from E3/19K in being unable to prevent the presentation of Kd-restricted viral proteins to CTLs. This is unlikely to be due to delta E19 having a lower avidity for Kd than E3/19K, since delta E19 was able to compete with E3/19K for Kd binding, both physically, and functionally in nullifying the E3/19K blockade of antigen presentation. These findings indicate that the ability of E3/19K to block antigen presentation is due solely to its ability to retain newly synthesized class I molecules in the endoplasmic reticulum.

Effect of various inhibitors on sporulation of Bacillus subtilis crs mutants

1984 ◽  
Vol 30 (11) ◽  
pp. 1337-1343 ◽  
Author(s):  
I. Takahashi ◽  
Dongxu Sun
Keyword(s):  
Bacillus Subtilis ◽  
Microscopic Examination ◽  
Electron Microscopic Examination ◽  
Aliphatic Alcohols ◽  
Wild Type ◽  
Electron Microscopic

The effect of aliphatic alcohols, cerulenin, and NaCl on sporulation of various catabolite-resistant (crs) mutants of Bacillus subtilis was studied. Mutants carrying crsA or crsF mutations were able to sporulate in the presence of these agents. Other crs mutants were resistant to at least one of the inhibitors. Electron microscopic examination revealed that cerulenin blocks sporulation at stage 0 in wild-type cells, suggesting that early sporulation functions are affected by this antibiotic. The results obtained so far suggest that the functions altered in the crs mutants may be related to the membrane.

scholarly journals Smallpox vaccines induce antibodies to the immunomodulatory, secreted vaccinia virus complement control protein

2009 ◽  
Vol 90 (11) ◽  
pp. 2604-2608 ◽  
Author(s):  
Joan E. Adamo ◽  
Clement A. Meseda ◽  
Jerry P. Weir ◽  
Michael J. Merchlinsky
Keyword(s):  
Vaccinia Virus ◽  
Humoral Response ◽  
Viral Proteins ◽  
Secreted Proteins ◽  
Complement Protein ◽  
Lethal Challenge ◽  
Complement Control Protein ◽  
Infected Cells ◽  
Control Protein ◽  
Primary Band

Vaccination with Dryvax elicits a broad humoral response against many viral proteins. Human vaccinia immune globulin was used to screen the secreted proteins from cells infected with Dryvax or the candidate smallpox vaccine LC16m8 to determine whether the protective humoral response included antibodies against secreted viral proteins. Many proteins were detected, with the primary band corresponding to a band of 28 or 30 kDa in cells infected with Dryvax or LC16m8, respectively. This was identified as the vaccinia virus complement protein (VCP), which migrated more slowly in LC16m8-infected cells due to post-translational glycosylation. Vaccinia virus deleted in VCP, vVCPko, protected mice from a lethal intranasal challenge of vaccinia Western Reserve strain. Mice vaccinated with purified VCP demonstrated a strong humoral response, but were not protected against a moderate lethal challenge of vaccinia virus, suggesting that the humoral response against VCP is not critical for protection.

scholarly journals Characterization of the Vaccinia Virus A35R Protein and Its Role in Virulence

2006 ◽  
Vol 80 (1) ◽  
pp. 306-313 ◽  
Author(s):  
Rachel L. Roper
Keyword(s):  
Vaccinia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Growth ◽  
Protein Band ◽  
Mutant Virus ◽  
Translation System ◽  
Wild Type ◽  
Infected Cells

ABSTRACT The vaccinia virus A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidic protein. Western blotting with anti-A35R peptide antibodies indicated that the protein is expressed early in infection and resolved as a single sharp band of ∼23 kDa, slightly higher than the 20 kDa predicted from its sequence. The protein band appeared to be the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whether expressed in an in vitro transcription/translation system without microsomes or expressed in infected cells, suggesting that it was not glycosylated. A mutant virus with the A35R gene deleted (vA35Δ) formed wild-type-sized plaques on all cell lines tested (human, monkey, mouse, and rabbit); thus, A35R is not required for replication and does not appear to be a host range gene. Although the A35R protein is hydrophobic, it is unlikely to be an integral membrane protein, as it partitioned to the aqueous phase during TX-114 partitioning. The protein could not be detected in virus-infected cell supernatants. A35R localized intracellularly to the virus factories, where the first stages of morphogenesis occur. The vA35Δ mutant formed near-normal levels of the various morphogenic stages of infectious virus particles and supported normal acid-induced fusion of virus-infected cells. Despite normal growth and morphogenesis in vitro, the vA35Δ mutant virus was attenuated in intranasal challenge of mice compared to wild-type and A35R rescue virus. Thus, the intracellular A35R protein plays a role in virulence. The A35R has little homology to any protein outside of poxviruses, suggesting a novel virulence mechanism.

scholarly journals The SARS CoV-1 3a protein disrupts Golgi complex morphology and cargo trafficking

2021 ◽  
Author(s):  
Rex R. Gonzales ◽  
Carolyn E. Machamer
Keyword(s):  
Infectious Bronchitis Virus ◽  
Viral Proteins ◽  
Transfected Cells ◽  
Infectious Bronchitis ◽  
E Protein ◽  
Complex Morphology ◽  
Universal Feature ◽  
Infected Cells ◽  
Intermediate Compartment ◽  
Acidic Compartments

Coronaviruses assemble by budding into the endoplasmic reticulum-Golgi intermediate compartment, but the pathway of egress from infected cells is not well understood. Efficient egress of infectious bronchitis virus (a gamma coronavirus, CoV) requires neutralization of Golgi pH by the envelope (E) protein. This results in reduced rates of cargo traffic and disrupts Golgi morphology, but it protects the spike protein from aberrant proteolysis. The severe acute respiratory syndrome (SARS) CoV-1 E protein does not disrupt the Golgi, however. We show here that in transfected cells, the ORF3a protein of SARS CoV-1 disrupts Golgi morphology, cargo trafficking and luminal pH. Unlike the infectious bronchitis virus E protein, these functions of the SARS CoV-1 3a protein appear to require its viroporin activity. Thus, neutralization of acidic compartments may be a universal feature of CoV infection, although different viral proteins and mechanisms may be used to achieve this outcome.

scholarly journals Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions

2012 ◽  
Vol 93 (9) ◽  
pp. 1876-1886 ◽  
Author(s):  
Virginie Doceul ◽  
Michael Hollinshead ◽  
Adrien Breiman ◽  
Kathlyn Laval ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Actin Polymerization ◽  
Protein S ◽  
Viral Proteins ◽  
Plaque Size ◽  
Cell Monolayers ◽  
Enveloped Virus ◽  
Short Consensus Repeat ◽  
Infected Cells ◽  
Mature Virus

Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

scholarly journals Genetic Analysis of the Vaccinia Virus I6 Telomere-Binding Protein Uncovers a Key Role in Genome Encapsidation

2003 ◽  
Vol 77 (20) ◽  
pp. 10929-10942 ◽  
Author(s):  
Olivera Grubisha ◽  
Paula Traktman
Keyword(s):  
Vaccinia Virus ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Electron Microscopic ◽  
Recombinant Viruses ◽  
Telomere Binding Protein ◽  
Microscopic Studies ◽  
Infected Cells ◽  
Genome Encapsidation

ABSTRACT The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.

scholarly journals Localization at high resolution of antibody-induced mobilization of vaccinia virus hemagglutinin and the major histocompatibility antigens on the plasma membrane of infected cells.

1982 ◽  
Vol 156 (5) ◽  
pp. 1435-1447 ◽  
Author(s):  
S Dales ◽  
M B Oldstone
Keyword(s):  
Light Microscopy ◽  
Vaccinia Virus ◽  
Cytotoxic T Lymphocyte ◽  
Electron Microscopic Examination ◽  
Major Histocompatibility ◽  
Electron Microscopic ◽  
Mhc Molecules ◽  
Major Histocompatibility Antigens ◽  
Gold Conjugate ◽  
20 Nm

We examined the consequence of simultaneous or independent binding of monospecific antibody to the hemagglutinin (HA) of vaccinia virus and the A-, B- and -determinants of HLA on HeLa or Raji cells or KkDk determinants of H-2 on L929 cells. The bound antibodies were marked by goat-anti-mouse (GAM) or goat-anti-rabbit (GAR) fluorochrome conjugates suitable for light microscopy and GAM or GAR gold conjugates, used in electron microscopy. Specificity and amount of antibody adsorbed was ascertained by complement-mediated lysis of 51Cr-labeled cells and by fluorescence-activated cell sorter analysis. Regardless of the order of either antibody to major histocompatibility complex (MHC) or antibody to HA addition after warming to 37 degrees C, there was evidence by light microscopy for co-patching and co-capping of the viral and host antigens. Electron microscopic examination revealed that goat-anti-rabbit 20 nM gold conjugate and goat-anti-mouse 5 nM gold conjugate, marking respectively the HA and MHC molecules, became concentrated in patched or caps in which the two antigens frequently overlapped or were closely associated. The contiguous MHC and HA antigens were also engulfed, as evidenced from the of two sizes of gold particles inside endocytic vacuoles. The significance of these observations is discussed in relation to the cytotoxic T lymphocyte-mediated killing virus-infected targets.

scholarly journals Vaccinia Virus A6 Is a Two-Domain Protein Requiring a Cognate N-Terminal Domain for Full Viral Membrane Assembly Activity

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Xiangzhi Meng ◽  
Lloyd Rose ◽  
Yue Han ◽  
Junpeng Deng ◽  
Yan Xiang
Keyword(s):  
Vaccinia Virus ◽  
Genetic Manipulation ◽  
Domain Architecture ◽  
Limited Proteolysis ◽  
Experimental System ◽  
Viral Proteins ◽  
Crescent Formation ◽  
Virion Assembly ◽  
Multistep Process ◽  
And Function

ABSTRACT Poxvirus virion biogenesis is a complex, multistep process, starting with the formation of crescent-shaped viral membranes, followed by their enclosure of the viral core to form spherical immature virions. Crescent formation requires a group of proteins that are highly conserved among poxviruses, including A6 and A11 of vaccinia virus (VACV). To gain a better understanding of the molecular function of A6, we established a HeLa cell line that inducibly expressed VACV-A6, which allowed us to construct VACV mutants with an A6 deletion or mutation. As expected, the A6 deletion mutant of VACV failed to replicate in noncomplementing cell lines with defects in crescent formation and A11 localization. Surprisingly, a VACV mutant that had A6 replaced with a close ortholog from the Yaba-like disease virus YLDV-97 also failed to replicate. This mutant, however, developed crescents and had normal A11 localization despite failing to form immature virions. Limited proteolysis of the recombinant A6 protein identified an N domain and a C domain of approximately 121 and 251 residues, respectively. Various chimeras of VACV-A6 and YLDV-97 were constructed, but only one that precisely combined the N domain of VACV-A6 and the C domain of YLDV-97 supported VACV replication albeit at a reduced efficiency. Our results show that VACV-A6 has a two-domain architecture and functions in both crescent formation and its enclosure to form immature virions. While a cognate N domain is not required for crescent formation, it is required for virion formation, suggesting that interactions of the N domain with cognate viral proteins may be critical for virion assembly. IMPORTANCE Poxviruses are unique among enveloped viruses in that they acquire their primary envelope not through budding from cellular membranes but by forming and extending crescent membranes. The crescents are highly unusual, open-ended membranes, and their origin and biogenesis have perplexed virologists for decades. A group of five viral proteins were recently identified as being essential for crescent formation, including the A6 protein of vaccinia virus. It is thus important to understand the structure and function of A6 in order to solve the long-standing mystery of poxvirus membrane biogenesis. Here, we established an experimental system that allowed the genetic manipulation of the essential A6L gene. By studying A6 mutant viruses, we found that A6 plays an essential role not only in the formation of crescents but also in their subsequent enclosure to form immature virions. We defined the domain architecture of A6 and suggested that one of its two domains cooperates with cognate viral proteins.

scholarly journals Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

2006 ◽  
Vol 80 (2) ◽  
pp. 553-561 ◽  
Author(s):  
Susan Parrish ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Deletion Mutant ◽  
Negative Regulator ◽  
Wild Type Virus ◽  
Wild Type ◽  
Gene Encoding ◽  
Early Proteins ◽  
Late Transcription ◽  
Infected Cells ◽  
Virus Mutant

ABSTRACT The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vΔD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vΔD9). Purified vΔD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vΔD9 virions. When 4 PFU per cell of wild-type or vΔD9 virions or equal numbers of vΔD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vΔD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vΔD10-infected cells than in cells infected with the wild type or vΔD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vΔD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.

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