scholarly journals Inactivation of hantaviruses by N-ethylmaleimide preserves virion integrity

2011 ◽  
Vol 92 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Tomas Strandin ◽  
Jussi Hepojoki ◽  
Hao Wang ◽  
Antti Vaheri ◽  
Hilkka Lankinen
Keyword(s):  
Cell Attachment ◽  
Buoyant Density ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Thiol Groups ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Nitrobenzoic Acid ◽  
The Family

Thiol groups of cysteine residues are crucial for the infectivity of various enveloped viruses, but their role in the infectivity of viruses of the family Bunyaviridae has thus far not been studied. This report shows that thiol groups are essential to the infectivity of hantaviruses. Alkylation of the thiol functional groups using the membrane-permeable compound N-ethylmaleimide (NEM) and membrane-impermeable compound 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) showed NEM to be a highly effective inactivator of Puumala and Tula hantaviruses. The NEM-inactivated hantavirus maintained the buoyant density of the wild-type virus. Furthermore, the antigenicity of glycoproteins and the cell attachment capacity of virions were retained at NEM concentrations that totally abolished virus infectivity. These results signified preservation of virion integrity following inactivation with NEM, making chemically inactivated virions valuable research antigens. It was demonstrated with biotin-conjugated maleimide, a mechanistic analogue of NEM, that all the structural proteins of hantavirus were sensitive towards thiol alkylation. In contrast to hantaviruses, NEM did not abolish Uukuniemi phlebovirus infectivity to the same extent. This indicates differences in the use of free thiols in virus entry among members of the family Bunyaviridae.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

2009 ◽  
Vol 83 (18) ◽  
pp. 9079-9093 ◽  
Author(s):  
Laura Pacini ◽  
Rita Graziani ◽  
Linda Bartholomew ◽  
Raffaele De Francesco ◽  
Giacomo Paonessa
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Line ◽  
Genome Structure ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Wild Type ◽  
Naturally Occurring

ABSTRACT Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

scholarly journals Role of Herpes Simplex Virus ICP27 in the Degradation of mRNA by Virion Host Shutoff RNase

2010 ◽  
Vol 84 (19) ◽  
pp. 10182-10190 ◽  
Author(s):  
Brunella Taddeo ◽  
Weiran Zhang ◽  
Bernard Roizman
Keyword(s):  
Mutant Virus ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Tegument Protein ◽  
Wild Type ◽  
Virion Host Shutoff ◽  
Infected Cells ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT The virion host shutoff (VHS) RNase tegument protein released into cells by infecting virus has two effects. Preexisting stable mRNAs (e.g., GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) are rapidly degraded. Stress response RNAs containing AU-rich elements (AREs) in the 3′ untranslated region (3′UTR) are deadenylated and cleaved, but the cleavage products persist for hours, in contrast to the short half-lives of ARE-containing mRNAs in uninfected cells. At late times, the VHS RNase is neutralized by the viral structural proteins VP16 and VP22. A recent study (J. A. Corcoran, W. L. Hsu, and J. R. Smiley, J. Virol. 80:9720-9729, 2006) reported that, at relatively late times after infection, ARE RNAs are rapidly degraded in cells infected with ΔICP27 mutant virus and concluded that ICP27 “stabilizes” ARE mRNAs. We report the following. (i) The rates of degradation of ARE mRNA at early times (3 h) after infection with the wild type or the ΔICP27 mutant virus are virtually identical, and hence ICP27 plays no role in this process. (ii) In noncomplementing cells, VHS RNase or VP22 is not synthesized. Therefore, the only VHS that is active is brought into cells by the ΔICP27 mutant. (ii) The VHS RNase brought into the cells by the ΔICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in ΔICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in ΔVHS RNase mutant-virus-infected cells and does not by itself support the hypothesis that ICP27 stabilizes ARE mRNAs. (iii) Concurrently, we present the first evidence that VHS RNase interacts with ICP27 most likely when bound to cap- and poly(A)-binding proteins, respectively.

scholarly journals The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

2021 ◽  
Vol 17 (8) ◽  
pp. e1009803
Author(s):  
Dipanwita Mitra ◽  
Mohammad H. Hasan ◽  
John T. Bates ◽  
Michael A. Bierdeman ◽  
Dallas R. Ederer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Virus Entry ◽  
Degree Of Polymerization ◽  
Host Cells ◽  
Virus Infectivity ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Host Membrane ◽  
Mutant Cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

scholarly journals Glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

2021 ◽  
Author(s):  
Shijian Zhang ◽  
Eden P. Go ◽  
Haitao Ding ◽  
Saumya Anang ◽  
John C. Kappes ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Etiologic Agent ◽  
Host Cells ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Wild Type ◽  
Spike Glycoprotein ◽  
Sugar Addition

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by co-expression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to purify the Golgi-modified fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on other characterized S trimer preparations, is predominantly modified in the Golgi compartment by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions. IMPORTANCE The SARS-CoV-2 coronavirus, which causes COVID-19, uses its spike glycoprotein to enter host cells. The viral spike glycoprotein is the main target of host neutralizing antibodies that help to control SARS-CoV-2 infection and are important for the protection provided by vaccines. The SARS-CoV-2 spike glycoprotein consists of a trimer of two subunits covered with a coat of carbohydrates (sugars). Here, we describe the disulfide bonds that assist the SARS-CoV-2 spike glycoprotein to assume the correct shape, and the composition of the sugar moieties on the glycoprotein surface. We also evaluate the consequences of natural virus variation in O-linked sugar addition and in the cysteine residues involved in disulfide bond formation. This information can expedite the improvement of vaccines and therapies for COVID-19.

scholarly journals Cleavage of Human Cytomegalovirus Protease pUL80a at Internal and Cryptic Sites Is Not Essential but Enhances Infectivity

2005 ◽  
Vol 79 (20) ◽  
pp. 12961-12968 ◽  
Author(s):  
Amy N. Loveland ◽  
Chee-Kai Chan ◽  
Edward J. Brignole ◽  
Wade Gibson
Keyword(s):  
Human Cytomegalovirus ◽  
Cleavage Site ◽  
Biological Significance ◽  
Virus Production ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Biological Importance

ABSTRACT The cytomegalovirus (CMV) maturational protease, assemblin, contains an “internal” (I) cleavage site absent from its homologs in other herpesviruses. Blocking this site for cleavage did not prevent replication of the resulting I− mutant virus. However, cells infected with the I− virus showed increased amounts of a fragment produced by cleavage at the nearby “cryptic” (C) site, suggesting that its replication may bypass the I-site block by using the C site as an alternate cleavage pathway. To test this and further examine the biological importance of these cleavages, we constructed two additional virus mutants—one blocked for C-site cleavage and another blocked for both I- and C-site cleavage. Infectivity comparisons with the parental wild-type virus showed that the I− mutant was the least affected for virus production, whereas infectivity of the C− mutant was reduced by ≈40% and when both sites were blocked virus infectivity was reduced by nearly 90%, providing the first evidence that these cleavages have biological significance. We also present and discuss evidence suggesting that I-site cleavage destabilizes assemblin and its fragments, whereas C-site cleavage does not.

Cryo-Electron Microscopy of Aura Viruses

1998 ◽  
Vol 4 (S2) ◽  
pp. 946-947
Author(s):  
W. Zhang ◽  
N. H. Olson ◽  
B. R. McKinney ◽  
R. J. Kuhn ◽  
T. S. Baker
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Single Copy ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Subgenomic Rna ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Cryo Electron Microscopy

Alphaviruses are a group of enveloped viruses in the Togaviridae family. Studies of several alphaviruses, including Ross River, Sindbis and Semliki Forest viruses, by cryo-electron microscopy (cryo-EM), three-dimensional (3D) image resconstruction and other techniques have illustrated that these spherical viruses have a T=4, multi-layered structure.Aura virus, which is closely related to Sindbis, was first isolated in South America. Unlike the other alphaviruses, both genomic RNA (12kb, 49S) and subgenomic RNA(4.2kb, 26S) are encapsidated efficiently and form mature virions. Studies on negatively-stained virus particles demonstrated that there are two major size classes. The first contains particles of ∼72nm diameter, which are most similar to wild type virus, whereas the second class includes particles of ∼62nm in diameter. The 72nm particles are believed to have one copy of genomic RNA or one to three copies of subgenomic RNA, and a T=4 structure. The 62nm particles probably only have a single copy of subgenomic RNA and are presumed to be T=3 structures.

scholarly journals Human Rhinovirus Type 16: Mutant V1210A Requires Capsid-Binding Drug for Assembly of Pentamers To Form Virions during Morphogenesis

2003 ◽  
Vol 77 (11) ◽  
pp. 6235-6244 ◽  
Author(s):  
Wai-Ming Lee ◽  
Wensheng Wang
Keyword(s):  
Cdna Clone ◽  
Virus Assembly ◽  
Culture Collection ◽  
Human Rhinovirus ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Virus Infectivity ◽  
Wild Type ◽  
Drug Dependent

ABSTRACT Our laboratory has previously reported isolation of human rhinovirus type 16 (HRV16) mutants which depend on WIN 52035 to grow. A rapid rise of progeny virus infectivity occurred when drug was added late in growth cycles, suggesting that the drug-dependence lesion was at the step of virus assembly (W. Wang et al., J. Virol. 72:1210-1218, 1998). Here, we report that capsid subunits, 5S protomers and 14S pentamers, of a drug-dependent mutant were produced normally in the absence of drug, but mutant 80S empty capsids and 150S provirions were not formed, maturation cleavage of provirions (VP0 → VP2 + VP4) did not occur, and the unassembled mutant capsid subunits were degraded with a half-life of 15 min. Drug was not required by mutant virus for attachment, uncoating, RNA synthesis and protein synthesis, and polyprotein processing except maturation cleavage. The requirement of drug for assembly of mutant pentamers to form provirions and the rapid assembly of preformed subunits (synthesized in the absence of drug) after drug addition suggested that after native pentamers (P5) have been formed they must be converted to an assembly active state (P5∗), possibly by a conformational change induced by the binding of drug. We propose that pocket factor plays the same role in wild-type virus. In addition, we also report the construction and the properties of a full-length cDNA clone of HRV16, pR16.11, which produces in vitro transcripts with infectivity similar to that of virion RNA. This cDNA clone is available at the American Type Culture Collection.

scholarly journals Antigenic differences between wildtype measles viruses and vaccine viruses in Indonesia

2016 ◽  
Vol 48 (3) ◽  
pp. 125
Author(s):  
Made Setiawan ◽  
Agus Sjahrurachman ◽  
Fera Ibrahim ◽  
Agus Suwandono
Keyword(s):  
Measles Virus ◽  
Neutralization Test ◽  
Vaccine Virus ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Type Virus ◽  
Measles Vaccine ◽  
Wild Type ◽  
Examination Technique ◽  
Cross Neutralization

Background Measles virus has a single, negative strand RNAgenome which codes 6 structural proteins: N, F, P M, H and L.Currently there are several variances in the nucleotide sequencesof N, F, M and H genes across wild type measles viruses, hencemeasles viruses can be categorized into clades and genotypes. Theantigenicity of the previous genotype of measles is different fromthe current genotype.Objective To determine the antigenic differences between wildtype measles virus and measles vaccine virus.Methods Analysis of the antigenic differences between wild typevirus (G2, G3 and D9) and vaccine virus (CAM-70 and Schwarz)was performed by immunizing mice with the respective viruses.The serum was then tested with micro-cross-neutralizationtechnique using the G2, G3, D9 and CAM-70 virus. Tests withcross ELISA examination technique were also performed usingthe same set of virus.Results Analysis of the cross neutralization test and cross ELISAshowed that the highest antigenicity reaction was found betweenwild type virus with antibody against wild type virus, while thelowest reaction was between wild type virus with antibody againstCAM-70.Conclusions We conclude that the antigenicity of antigenic proteinfrom wild type virus is higher than antigenicity of vaccine virusprotein. In addition, it was found that the antigenicity of proteinsfrom Schwarz vaccine virus was higher than proteins CAM-70vaccine virus.

scholarly journals Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

2021 ◽  
Author(s):  
Shijian Zhang ◽  
Eden P. Go ◽  
Haitao Ding ◽  
Saumya Anang ◽  
John C. Kappes ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Etiologic Agent ◽  
Host Cells ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Wild Type ◽  
Disulfide Bonding ◽  
Natural Variants ◽  
Complex Forms

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.

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