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1.
Sci Rep ; 11(1): 14965, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294758

RESUMO

The TRDC-locus encodes the T cell receptor delta constant region, one component of the γδ T cell receptor which is essential for development of γδ T cells. In contrast to peptide recognition by αß T cells, antigens activating γδ T cells are mostly MHC independent and not well characterized. Therefore, the function of γδ T cells and their contribution to protection against infections is still unclear. Higher numbers of circulating γδ T cells compared to mice, render the pig a suitable animal model to study γδ T cells. Knocking-out the porcine TRDC-locus by intracytoplasmic microinjection and somatic cell nuclear transfer resulted in healthy living γδ T cell deficient offspring. Flow cytometric analysis revealed that TRDC-KO pigs lack γδ T cells in peripheral blood mononuclear cells (PBMC) and spleen cells. The composition of the remaining leucocyte subpopulations was not affected by the depletion of γδ T cells. Genome-wide transcriptome analyses in PBMC revealed a pattern of changes reflecting the impairment of known or expected γδ T cell dependent pathways. Histopathology did not reveal developmental abnormalities of secondary lymphoid tissues. However, in a vaccination experiment the KO pigs stayed healthy but had a significantly lower neutralizing antibody titer as the syngenic controls.

2.
Viruses ; 13(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201636

RESUMO

Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor.

3.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298900

RESUMO

Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.


Assuntos
Pestivirus/genética , Proteínas do Envelope Viral/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Dimerização , Mutação/genética , Coelhos , Internalização do Vírus
4.
J Virol ; 95(15): e0052121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011544

RESUMO

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2. IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.


Assuntos
Vírus da Diarreia Viral Bovina/metabolismo , Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Vírus da Diarreia Viral Bovina/genética , Glicoproteínas de Membrana/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Coelhos , Proteínas do Envelope Viral/genética
5.
Viruses ; 13(3)2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801849

RESUMO

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.


Assuntos
Sequência de Aminoácidos/genética , Potenciais da Membrana/genética , Pestivirus/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Galinhas , Cricetinae , Genoma Viral/genética , Glicosilação , Proteínas de Membrana/metabolismo , Mutação/genética , Pestivirus/genética , Serina Endopeptidases/metabolismo , Carga Viral , Fatores de Virulência/genética
6.
J Virol ; 95(1)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33028718

RESUMO

Like other enveloped viruses, pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxy terminus of the signal sequence preceding Erns and the E1/E2 and E2/P7 sites, the Erns-E1 precursor is cleaved by signal peptidase at a highly unusual structure, in which the transmembrane sequence upstream of the cleavage site is replaced by an amphipathic helix. As shown before, the integrity of the amphipathic helix is crucial for efficient processing. The data presented here demonstrate that the E1 sequence downstream of this cleavage site is also important for the cleavage. Carboxy-terminal truncation of the E1 moiety as well as internal deletions in E1 reduced the cleavage efficiency to less than 30% of the wild-type (wt) level. Moreover, the C-terminal truncation by more than 30 amino acids resulted in strong secretion of the uncleaved fusion proteins. The reduced processing and increased secretion were even observed when 10 to 5 amino-terminal residues of E1 were left, whereas extensions by 1 or 3 E1 residues resulted in reduced processing but no significantly increased secretion. In contrast to the E1 sequences, a 10-amino-acid c-myc tag fused to the Erns C terminus had only marginal effect on secretion but was also not processed efficiently. Mutation of the von Heijne sequence upstream of E2 not only blocked the cleavage between E1 and E2 but also prevented the processing between Erns and E2. Thus, processing at the Erns-E1 site is a highly regulated process.IMPORTANCE Cellular signal peptidase (SPase) cleavage represents an important step in maturation of viral envelope proteins. Fine tuning of this system allows for establishment of concerted folding and processing processes in different enveloped viruses. We report here on SPase processing of the Erns-E1-E2 glycoprotein precursor of pestiviruses. Erns-E1 cleavage is delayed and only executed efficiently when the complete E1 sequence is present. C-terminal truncation of the Erns-E1 precursor impairs processing and leads to significant secretion of the protein. The latter is not detected when internal deletions preserving the E1 carboxy terminus are introduced, but also these constructs show impaired processing. Moreover, Erns-E1 is only processed after cleavage at the E1/E2 site. Thus, processing of the pestiviral glycoprotein precursor by SPase is done in an ordered way and depends on the integrity of the proteins for efficient cleavage. The functional importance of this processing scheme is discussed in the paper.


Assuntos
Pestivirus/metabolismo , Poliproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Proteínas de Membrana/metabolismo , Mutação , Poliproteínas/química , Poliproteínas/genética , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
7.
Nucleic Acids Res ; 47(4): 1920-1934, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30668745

RESUMO

Caliciviruses use a termination/reinitiation mechanism for translation of their minor capsid protein VP2. A sequence element of about 80 nucleotides denoted 'termination upstream ribosomal binding site' (TURBS) is crucial for reinitiation. RNA secondary structure probing and computer aided secondary structure prediction revealed a rather low degree of secondary structure determinants for the TURBS of the rabbit hermorrhagic disease virus. Mutation analysis showed that prevention of duplex formation had major impact on the VP2 expression levels. Restoration of complementarity of the respective sequences by reciprocal mutation at least partially restored reinitiating rates. Synthetic TURBS structures preserving only the secondary structure forming sequences and the known short motifs important for TURBS function were found to drive reinitiation when the altered sequence could be predicted to allow establishment of the crucial secondary structures of the TURBS.


Assuntos
Infecções por Caliciviridae/genética , Proteínas do Capsídeo/genética , Vírus da Doença Hemorrágica de Coelhos/genética , Relação Estrutura-Atividade , Animais , Sítios de Ligação , Infecções por Caliciviridae/virologia , Regulação Viral da Expressão Gênica/genética , Vírus da Doença Hemorrágica de Coelhos/patogenicidade , Mutação , Biossíntese de Proteínas/genética , Coelhos , Ribossomos/genética
8.
J Gen Virol ; 99(1): 86-96, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29235980

RESUMO

The classical swine fever virus (CSFV) represents one of the most important pathogens of swine. The CSFV glycoprotein Erns is an essential structural protein and an important virulence factor. The latter is dependent on the RNase activity of this envelope protein and, most likely, its secretion from the infected cell. A further important feature with regard to its function as a virulence factor is the formation of disulfide-linked Erns homodimers that are found in virus-infected cells and virions. Mutant CSFV lacking cysteine (Cys) 171, the residue responsible for intermolecular disulfide bond formation, were found to be attenuated in pigs (Tews BA, Schürmann EM, Meyers G. J Virol 2009;83:4823-4834). In the course of an animal experiment with such a dimerization-negative CSFV mutant, viruses were reisolated from pigs that contained a mutation of serine (Ser) 209 to Cys. This mutation restored the ability to form disulphide-linked Erns homodimers. In transient expression studies Erns mutants carrying the S209C change were found to form homodimers with about wt efficiency. Also the secretion level of the mutated proteins was equivalent to that of wt Erns. Virus mutants containing the Cys171Ser/Ser209Cys configuration exhibited wt growth rates and increased virulence when compared with the Cys171Ser mutant. These results provide further support for the connection between CSFV virulence and Erns dimerization.


Assuntos
Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/patogenicidade , Peste Suína Clássica/virologia , Células Epiteliais/virologia , Mutação , Proteínas do Envelope Viral/genética , Animais , Sequência de Bases , Linhagem Celular , Peste Suína Clássica/patologia , Vírus da Febre Suína Clássica/metabolismo , Cricetulus , Expressão Gênica , Engenharia Genética , Rim/virologia , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suínos , Proteínas do Envelope Viral/metabolismo , Carga Viral , Virulência
9.
J Gen Virol ; 98(8): 2106-2112, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28786787

RESUMO

We propose the creation of seven new species in the genus Pestivirus (family Flaviviridae) in addition to the four existing species, and naming species in a host-independent manner using the format Pestivirus X. Only the virus species names would change; virus isolates would still be referred to by their original names. The original species would be re-designated as Pestivirus A (original designation Bovine viral diarrhea virus 1), Pestivirus B (Bovine viral diarrhea virus 2), Pestivirus C (Classical swine fever virus) and Pestivirus D (Border disease virus). The seven new species (and example isolates) would be Pestivirus E (pronghorn pestivirus), Pestivirus F (Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H (Hobi-like pestivirus), Pestivirus I (Aydin-like pestivirus), Pestivirus J (rat pestivirus) and Pestivirus K (atypical porcine pestivirus). A bat-derived virus and pestiviruses identified from sheep and goat (Tunisian sheep pestiviruses), which lack complete coding region sequences, may represent two additional species.


Assuntos
Infecções por Pestivirus/veterinária , Pestivirus/classificação , Pestivirus/isolamento & purificação , Animais , Bovinos , Cabras , Pestivirus/genética , Pestivirus/fisiologia , Infecções por Pestivirus/virologia , Filogenia , Ratos , Ovinos , Suínos
10.
Virology ; 507: 123-134, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28432927

RESUMO

Bovine viral diarrhea virus (BVDV) infection induces immunosuppression and in conjunction with bovine respiratory syncytial virus (BRSV) contributes to the bovine respiratory disease complex. Bovine turbinate cells were single or co-infected with type 2 BVDV wild-type (BVDV2-wt), its dysfunctional Npro mutant (BVDV2-E), and/or BRSV. BVDV2-E significantly up-regulated PKR, IRF-7, TBK-1, IRF-3, and IFN-ß mRNAs based on real-time Q-RT-PCR. BRSV-infected cells expressed significantly up-regulated PKR, IRF-3, IRF-7, and IFN-ß mRNAs, whereas BVDV2-wt, but not BVDV2-E, abolished this up-regulation in co-infection. No significant differences were observed in MAVS, NF-κB, and PIN-1 mRNAs. A dual-luciferase reporter assay showed that BVDV2-wt significantly increased NF-κB activity compared to BVDV2-E, while BVDV2-E significantly increased IFN-ß activity compared to BVDV2-wt. The BRSV titer and RNA levels significantly increased in cells co-infected with BRSV/BVDV2-wt compared to cells co-infected with BRSV/BVDV2-E or infected with BRSV alone. This data supports the synergistic action of BVDV2-wt and BRSV inhibition of IFN-1.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/metabolismo , Vírus da Diarreia Viral Bovina Tipo 2/fisiologia , Interferon beta/metabolismo , Infecções por Vírus Respiratório Sincicial/veterinária , Transdução de Sinais , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/genética , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Coinfecção/genética , Coinfecção/imunologia , Coinfecção/virologia , Vírus da Diarreia Viral Bovina Tipo 2/genética , Interferon beta/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Bovino/genética , Vírus Sincicial Respiratório Bovino/fisiologia , Replicação Viral
11.
J Gen Virol ; 98(1): 2-3, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28218572

RESUMO

The Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000-13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens (e.g. yellow fever virus, dengue virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at www.ictv.global/report/flaviviridae.


Assuntos
Flaviviridae/classificação , Animais , Vetores Artrópodes/virologia , Flaviviridae/genética , Flaviviridae/fisiologia , Flaviviridae/ultraestrutura , Infecções por Flaviviridae/transmissão , Infecções por Flaviviridae/veterinária , Infecções por Flaviviridae/virologia , Humanos
12.
Methods Mol Biol ; 1499: 15-35, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27987141

RESUMO

Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.


Assuntos
Vírus de RNA/genética , Vírus de RNA/imunologia , RNA Viral/genética , RNA Viral/imunologia , Vacinas/imunologia , Animais , Humanos , Replicon/genética , Replicon/imunologia , Vacinas/genética
13.
J Gen Virol ; 97(11): 2894-2907, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27692039

RESUMO

Proposals are described for the assignment of recently reported viruses, infecting rodents, bats and other mammalian species, to new species within the Hepacivirus and Pegivirus genera (family Flaviviridae). Assignments into 14 Hepacivirus species (Hepacivirus A-N) and 11 Pegivirus species (Pegivirus A-K) are based on phylogenetic relationships and sequence distances between conserved regions extracted from complete coding sequences for members of each proposed taxon. We propose that the species Hepatitis C virus is renamed Hepacivirus C in order to acknowledge its unique historical position and so as to minimize confusion. Despite the newly documented genetic diversity of hepaciviruses and pegiviruses, members of these genera remain phylogenetically distinct, and differ in hepatotropism and the possession of a basic core protein; pegiviruses in general lack these features. However, other characteristics that were originally used to support their division into separate genera are no longer definitive; there is overlap between the two genera in the type of internal ribosomal entry site and the presence of miR-122 sites in the 5' UTR, the predicted number of N-linked glycosylation sites in the envelope E1 and E2 proteins, the presence of poly U tracts in the 3' UTR and the propensity of viruses to establish a persistent infection. While all classified hepaciviruses and pegiviruses have mammalian hosts, the recent description of a hepaci-/pegi-like virus from a shark and the likely existence of further homologues in other non-mammalian species indicate that further species or genera remain to be defined in the future.


Assuntos
Infecções por Flaviviridae/veterinária , Infecções por Flaviviridae/virologia , Flaviviridae/classificação , Hepacivirus/classificação , Hepatite C/veterinária , Hepatite C/virologia , Animais , Quirópteros/virologia , Flaviviridae/genética , Flaviviridae/isolamento & purificação , Variação Genética , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Humanos , Mamíferos/virologia , Filogenia , Roedores/virologia , Análise de Sequência de DNA
14.
J Immunol ; 196(10): 4214-26, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27053760

RESUMO

The pestivirus noncytopathic bovine viral diarrhea virus (BVDV) can suppress IFN production in the majority of cell types in vitro. However, IFN is detectable in serum during acute infection in vivo for ∼5-7 d, which correlates with a period of leucopoenia and immunosuppression. In this study, we demonstrate that a highly enriched population of bovine plasmacytoid dendritic cells (DCs) produced IFN in response to BVDV in vitro. We further show that the majority of the IFN produced in response to infection both in vitro and in vivo is type III IFN and acid labile. Further, we show IL-28B (IFN-λ3) mRNA is induced in this cell population in vitro. Supernatant from plasmacytoid DCs harvested postinfection with BVDV or recombinant bovine IFN-α or human IL-28B significantly reduced CD4(+) T cell proliferation induced by tubercle bacillus Ag 85-stimulated monocyte-derived DCs. Furthermore, these IFNs induced IFN-stimulated gene expression predominantly in monocyte-derived DCs. IFN-treated immature DCs derived from murine bone marrow also had a reduced capacity to stimulate T cell proliferative responses to tubercle bacillus Ag 85. Immature DCs derived from either source had a reduced capacity for Ag uptake following IFN treatment that is dose dependent. Immunosuppression is a feature of a number of pestivirus infections; our studies suggest type III IFN production plays a key role in the pathogenesis of this family of viruses. Overall, in a natural host, we have demonstrated a link between the induction of type I and III IFN after acute viral infection and transient immunosuppression.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Células Dendríticas/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Imunidade Celular , Interferon-alfa/imunologia , Interleucinas/imunologia , Aciltransferases/imunologia , Animais , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Bovinos , Linhagem Celular , Proliferação de Células , Humanos , Tolerância Imunológica , Interferon-alfa/sangue , Interferons , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/imunologia , Proteínas Recombinantes/imunologia , Sus scrofa
15.
PLoS One ; 10(8): e0135680, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26270479

RESUMO

Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.


Assuntos
Glicoproteínas/química , Lipídeos de Membrana/metabolismo , Pestivirus/metabolismo , Proteínas Virais/química , Animais , Sítios de Ligação , Linhagem Celular , Cricetinae , Glicoproteínas/genética , Glicoproteínas/metabolismo , Mutação , Pestivirus/química , Pestivirus/genética , Estrutura Secundária de Proteína , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
Adv Virus Res ; 93: 47-160, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26111586

RESUMO

Pestiviruses are among the economically most important pathogens of livestock. The biology of these viruses is characterized by unique and interesting features that are both crucial for their success as pathogens and challenging from a scientific point of view. Elucidation of these features at the molecular level has made striking progress during recent years. The analyses revealed that major aspects of pestivirus biology show significant similarity to the biology of human hepatitis C virus (HCV). The detailed molecular analyses conducted for pestiviruses and HCV supported and complemented each other during the last three decades resulting in elucidation of the functions of viral proteins and RNA elements in replication and virus-host interaction. For pestiviruses, the analyses also helped to shed light on the molecular basis of persistent infection, a special strategy these viruses have evolved to be maintained within their host population. The results of these investigations are summarized in this chapter.


Assuntos
Infecções por Pestivirus/veterinária , Pestivirus/genética , Animais , Genoma Viral , Humanos , Gado , Pestivirus/classificação , Pestivirus/isolamento & purificação , Infecções por Pestivirus/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
PLoS One ; 9(7): e102254, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25007260

RESUMO

Caliciviruses use reinitiation of translation governed by a 'termination upstream ribosomal binding site' (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.


Assuntos
Caliciviridae/fisiologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , RNA Mensageiro/metabolismo , Animais , Caliciviridae/genética , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Gatos , Linhagem Celular , Cricetinae , Conformação de Ácido Nucleico , RNA Viral/metabolismo , Replicação Viral
18.
PLoS Pathog ; 10(2): e1003973, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24586172

RESUMO

E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns) membrane contact, processing and secretion.


Assuntos
Pestivirus/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína
19.
J Biol Chem ; 289(17): 11739-11754, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24599949

RESUMO

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.


Assuntos
Norovirus/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Códon , Cricetinae , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Ribossômico 18S/genética , Regiões Terminadoras Genéticas , Proteínas Virais/genética
20.
FASEB J ; 26(8): 3292-305, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22549508

RESUMO

Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals. Secretion of the protein is regarded as crucial for its function as a viral virulence factor associated with its RNase activity. However, ∼95% of the E(rns) molecules are retained within the infected cell. Fusion of different E(rns) fragments to the C terminus of CD72 allowed identification of a retention signal within the C-terminal 65 aa of the viral protein. This C-terminal sequence represents its membrane anchor and folds into an amphipathic helix binding in-plane to the membrane surface. Residues L183, I190, and L208 are important for intracellular location of E(rns). Presentation of the retention signal on the cytoplasmic instead of the luminal face of the ER membrane in CD8α fusion proteins still led to retention. Thus, E(rns) contains in its C-terminal amphipathic helix an intracellular retention signal that is active on both faces of the membrane.


Assuntos
Glicoproteínas de Membrana/química , Pestivirus/genética , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Células Cultivadas , Vírus da Febre Suína Clássica/genética , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
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