- Volume 89, Issue 3, 2008
Volume 89, Issue 3, 2008
- Review
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New insights into internal ribosome entry site elements relevant for viral gene expression
More LessA distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 5′ UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA–RNA contacts between the 5′ and 3′ ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.
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- Animal
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- RNA viruses
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West Nile 25A virus infection of B-cell-deficient (μMT) mice: characterization of neuroinvasiveness and pseudoreversion of the viral envelope protein
The attenuated West Nile virus 25A strain (WN25A) was investigated for its neuroinvasive properties in B-cell-deficient (μMT) mice. After peripheral inoculation, WN25A caused fatal encephalitis in the majority of 6–8-week-old mice, characterized by a systemic infection with viraemia, moderate virus burdens in peripheral tissues and a high titre of brain-associated virus. Mice generally succumbed to infection within a few weeks of infection. However, others survived for as long as 10 weeks, and some for even longer. Normal age-matched C57BL/6 mice showed no signs of illness after inoculation with WN25A virus. Nucleotide sequencing of WN25A viruses recovered from the brains of B-cell-deficient mice revealed that the conserved N-linked glycosylation site in the viral envelope protein was abolished by substitution of a serine residue at position 155. This was found to be a pseudoreversion relative to the wild-type WN-Israel strain, based on virulence testing of one such brain-associated virus in both B-cell-deficient and normal C57BL/6 mice. This study provides further characterization of the mouse virulence properties of the attenuated WN25A virus in the context of B-cell deficiency. Replication in these mice does not involve rapid neuroadaptation or reversion of WN25A virus to a neuroinvasive phenotype. Molecular modelling studies suggest a difference in local structure of the E protein associated with either an asparagine or serine residue at position 155 compared with the tyrosine found in the virulent parental WN-Israel virus.
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Structure-based mutagenesis identifies important novel determinants of the NS2B cofactor of the West Nile virus two-component NS2B–NS3 proteinase
West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B–NS3pro structure, we hypothesized that the G22 residue and the negatively charged patch D32DD34 of NS2B were part of an important configuration required for NS2B–NS3pro activity. Our experimental data confirmed that G22 and D32DD34 substitution for S and AAA, respectively, inactivated NS2B–NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B–NS3pro. Because of the significant level of homology in flaviviral NS2B–NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.
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Peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with downregulation of vascular endothelial cadherin
More LessPlasma leakage is one of the characteristic features of dengue haemorrhagic fever. The interaction among peripheral blood mononuclear cells (PBMCs), dengue virus and endothelial cells was analysed in vitro. Human umbilical vein endothelial cells (HUVECs) were infected with dengue-2 virus (DV-2) at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added to DV-2-infected HUVECs, and transendothelial electrical resistance (TEER) and transalbumin permeability were assessed. Dengue virus infection at an m.o.i. of 0.5 p.f.u. per cell alone did not decrease the TEER, but addition of PBMCs decreased the TEER, increased the albumin permeability and induced morphological changes of HUVECs. The extent of the decrease was more profound with adherent PBMCs than with non-adherent PBMCs. The expression of vascular endothelial cadherin (VE-cadherin) was examined using real-time RT-PCR and immunofluorescence. Addition of PBMCs to DV-2-infected HUVECs decreased the levels of mRNA transcripts and cell-surface expression of VE-cadherin. The results indicate that PBMCs increased the permeability of DV-2-infected HUVECs and that the increased permeability was concomitant with morphological change and the decrease in VE-cadherin expression. The results suggest that functional impairment of the DV-2-infected HUVEC monolayer was caused by interaction with PBMCs.
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Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein
The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1–6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.
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Abortions in dromedaries (Camelus dromedarius) caused by equine rhinitis A virus
A virus was isolated from aborted dromedary (Camelus dromedarius) fetuses during an abortion storm in Dubai, United Arab Emirates. Laboratory investigations showed the causative agent to be indistinguishable from equine rhinitis A virus (ERAV), a picornavirus. Two pregnant dromedaries experimentally infected with the camel virus isolate both aborted and an identical virus was reisolated from both fetuses, thus confirming the diagnosis. The extremely high prevalence of antibody (>90 %) and the high titres recorded against ERAV in the dromedary herd clearly showed that ERAV does infect dromedaries. Unlike horses, where ERAV targets the upper respiratory tract, in dromedaries the target organ appears to be the genital tract.
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An MHC-restricted CD8+ T-cell response is induced in cattle by foot-and-mouth disease virus (FMDV) infection and also following vaccination with inactivated FMDV
More LessFoot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that carries enormous economic consequences. CD8+ cytotoxic T lymphocytes play an important role in protection and disease outcome in viral infections but, to date, the role of the CD8+ T-cell immune response to FMDV remains unclear. This study aimed to investigate major histocompatibility complex (MHC) class I-restricted CD8+ T-cell responses to FMDV in vaccinated and in infected cattle. An in vitro assay was used to detect antigen-specific gamma interferon release by CD8+ T cells in FMDV-infected cattle of known MHC class I genotypes. A significant MHC class I-restricted CD8+ T-cell response was detected to both FMDV strain O1 BFS and a recombinant fowlpox virus expressing the structural proteins of FMDV. Antigen-specific MHC class I-restricted CD8+ T-cell responses were also detected in cattle vaccinated with inactivated FMDV. These responses were shown to be directed, at least in part, to epitopes within the structural proteins (P12A region) of the virus. By using mouse cells expressing single cattle MHC class I alleles, it was possible to identify the restriction elements in each case. Identification of these epitopes will facilitate the quantitative and qualitative analysis of FMDV-specific memory CD8+ T cells in cattle and help to ensure that potential vaccines induce a qualitatively appropriate CD8+ T-cell response.
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Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus
More LessThe cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.
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Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes
More LessWe previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection.
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H5N1 avian influenza re-emergence of Lake Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation
Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.
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Positively selected sites on the surface glycoprotein (G) of infectious hematopoietic necrosis virus
More LessMutations in the surface glycoprotein (G) of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that causes significant losses in hatcheries raising salmonid fish, were studied. A 303 nt segment (mid-G region) of this protein from 88 Idaho isolates of IHNV was sequenced. Evidence of positive selection at individual codon sites was estimated by using a Bayesian method (MrBayes). A software algorithm (CPHmodels) was used to construct a three-dimensional (3D) representation of the IHNV protein. The software identified structural homologies between the IHNV G protein and the surface glycoprotein of vesicular stomatitis virus (VSV) and used the VSV structure as a template for predicting the IHNV structure. The amino acids predicted to be under positive selection were mapped onto the proposed IHNV 3D structure and appeared at sites on the surface of the protein where antigen–antibody interaction should be possible. The sites identified as being under positive selection on the IHNV protein corresponded to those reported by others as active sites of mutation for IHNV, and also as antigenic sites on VSV. Knowledge of the sites where genetic variation is positively selected enables a better understanding of the interaction of the virus with its host, and with the host immune system. This information could be used to develop strategies for vaccine development for IHNV, as well as for other viruses.
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Feline immunodeficiency virus dendritic cell infection and transfer
More LessFeline immunodeficiency virus (FIV) interacts with dendritic cells (DC) during initiation of infection, but whether DC support or transfer FIV infection remains unclear. To address this issue, we studied the susceptibility of feline myeloid DC to FIV infection and assessed potential transfer of infection from DC to CD4+ T cells. FIV was detected in membrane-bound vesicles of DC within 2 h of inoculation, although only low concentrations of FIV DNA were found in virus-exposed isolated DC. Addition of resting CD4+ T cells increased viral DNA levels; however, addition of activated CD4+ T cells resulted in a burst of viral replication manifested by FIV p27 capsid antigen generation. To determine whether transfer of FIV infection required productively infected DC (vs virus bound to DC but not internalized), virus-exposed DC were cultured for 2 days to allow for degradation of uninternalized virus and initiation of infection in the DC, then CD4+ T blasts were added. Infection of T cells remained robust, indicating that T-cell infection is likely to be mediated by de novo viral infection of DC followed by viral transfer during normal DC/T-cell interactions. We conclude that feline DC support restricted FIV infection, which nevertheless is sufficient to efficiently transfer infection to susceptible T cells and trigger the major burst of viral replication. Feline DC/FIV/T-cell interactions (similar to those believed to occur in human immunodeficiency virus and simian immunodeficiency virus infections) highlight the means by which immunodeficiency-inducing lentiviruses exploit normal DC/T-cell interactions to transfer and amplify virus infection.
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Mutational analysis of a principal neutralization domain of visna/maedi virus envelope glycoprotein
We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site.
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APOBEC3G upregulation by alpha interferon restricts human immunodeficiency virus type 1 infection in human peripheral plasmacytoid dendritic cells
More LessAPOBEC3G (A3G), a member of cytidine deaminase family, has potent anti-human immunodeficiency virus type 1 (HIV-1) activity. It has been demonstrated that alpha interferon (IFN-α) can significantly enhance the expression of A3G in human primary resting CD4+ T-cells, macrophages and primary hepatocytes, subsequently decreasing their viral susceptibility. Plasmacytoid dendritic cells (pDCs) are key effectors in innate host immunity, mediating adaptive immune responses and stimulating IFN-α production in reaction to various stimuli. In this report, we demonstrate that IFN-α, either exogenously added to- or endogenously secreted by pDCs, can enhance the expression of A3G and its family members such as A3A, A3C and A3F. We have also shown that IFN-α can inhibit HIV-1 expression in pDCs. This inhibitory effect could be countered by addition of an A3G-specific short interfering RNA, indicating that IFN-α-induced A3G plays a key role in mediating pDCs response to HIV-1. Given the central role played by pDCs in orchestrating the IFN-α/A3G intercellular network and intracellular signal pathway, our data indicate that pDCs themselves are also protected by an IFN-α/A3G-mediated innate immunity barrier from HIV-1 infection.
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- DNA viruses
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Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53
A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50–pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.
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Recombinant simian varicella viruses expressing respiratory syncytial virus antigens are immunogenic
More LessRecombinant simian varicella viruses (rSVVs) were engineered to express respiratory syncytial virus (RSV) antigens. The RSV surface glycoprotein G and second matrix protein M2 (22k) genes were cloned into the SVV genome, and recombinant viruses were characterized in vitro and in vivo. rSVVs were also engineered to express the membrane-anchored or secreted forms of the RSV-G protein as well as an RSV G lacking its chemokine mimicry motif (CX3C), which may have different effects on priming the host immune response. The RSV genes were efficiently expressed in rSVV/RSV-infected Vero cells as RSV-G and -M2 transcripts were detected by RT-PCR, and RSV antigens were detected by immunofluorescence and immunoblot assays. The rSVVs replicated efficiently in Vero cell culture. Rhesus macaques immunized with rSVV/RSV-G and rSVV/RSV-M2 vaccines produced antibody responses to SVV and RSV antigens. The results demonstrate that recombinant varicella viruses are suitable vectors for the expression of RSV antigens and may represent a novel vaccine strategy for immunization against both pathogens.
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Primary naïve and interleukin-2-activated natural killer cells do not support efficient ectromelia virus replication
More LessNatural killer (NK) cells are known for their ability to lyse tumour cell targets. Studies of infections by a number of viruses, including poxviruses and herpesviruses, have demonstrated that NK cells are vital for recovery from these infections. Little is known of the ability of viruses to infect and complete a productive replication cycle within NK cells. Even less is known concerning the effect of infection on NK cell biology. This study investigated the ability of ectromelia virus (ECTV) to infect NK cells in vitro and in vivo. Following ECTV infection, NK cell gamma interferon (IFN-γ) production was diminished and infected cells ceased proliferating and lost viability. ECTV infection of NK cells led to early and late virus gene expression and visualization of immature and mature virus particles, but no detectable increase in viable progeny virus. It was not unexpected that early gene expression occurred in infected NK cells, as the complete early transcription system is packaged within the virions. The detection of the secreted early virus-encoded immunomodulatory proteins IFN-γ-binding protein and ectromelia inhibitor of complement enzymes (EMICE) in NK cell culture supernatants suggests that even semi-permissive infection may permit immunomodulation of the local environment.
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Porcine circovirus type 2-induced interleukin-10 modulates recall antigen responses
More LessPorcine circovirus type 2 (PCV2) is the necessary agent for the occurrence of post-weaning multisystemic wasting syndrome (PMWS) in pigs. It has been suggested that PMWS-affected pigs are immunosuppressed and, therefore, more prone to develop co-infections. In this study, we elucidated that PCV2 downregulates in vitro the immune cell functions during recall antigen responses. We showed that PCV2, but not the non-pathogenic porcine circovirus type 1, induces interleukin (IL)-10 secretion by monocytic cells. Notably, PCV2-induced IL-10 led to effective repression of IL-12 in blood peripheral mononuclear cells (PBMCs). Besides alpha and gamma interferon synthesis by PBMCs from pseudorabies virus (PRV)-immunized animals, activated in vitro PRV also was repressed by subsequent infection by PCV2. The ability of PCV2 to hamper the development of immune responses may contribute to the Th1 suppressed responses, immune suppression and co-infections.
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Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67
More LessOpen reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of ‘enveloped’ nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.
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Genomic sequence analysis of a fast-killing isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus
More LessSix clones of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were plaque-purified from field isolates collected in Missouri, USA. In bioassays, four of the plaque-purified isolates killed neonate S. frugiperda larvae more rapidly than the field isolates from which they were derived, with LT50 values (mean time to kill 50 % of the test larvae) ranging from 34.4 to 49.7 h post-infection. The complete genomic sequence of one of these isolates, SfMNPV-3AP2, was determined and analysed. The SfMNPV-3AP2 genome was 131 330 bp with a G+C content of 40.2 %. A total of 144 open reading frames (ORFs) was identified and examined, including the set of 62 genes in common among lepidopteran nucleopolyhedrovirus genomes. Comparisons of ORF content, order and predicted amino acid sequences with other nucleopolyhedoviruses indicated that SfMNPV is part of a cluster of viruses within NPV group II that includes NPVs isolated from Spodoptera, Agrotis and Mamestra host species. SfMNPV-3AP2 shared a high degree of nucleotide sequence similarity with partial sequences from other SfMNPV isolates. Comparison of the SfMNPV-3AP2 genome sequence with a partial sequence from a Brazilian isolate of SfMNPV revealed that SfMNPV-3AP2 contained a deletion that removed parts of ORF sf27 and the gene encoding ecdysteroid UDP-glucosyltransferase (egt). An examination of the egt region in the other isolates revealed that the other five SfMNPV clones also contained deletions of varying length in this region. Variant genotypes with deletions extending around the egt gene have been reported previously from a Nicaraguan field isolate of SfMNPV, suggesting that the presence of such variants is a common feature of SfMNPV populations.
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The F protein of Helicoverpa armigera single nucleopolyhedrovirus can be substituted functionally with its homologue from Spodoptera exigua multiple nucleopolyhedrovirus
More LessF proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacΔF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacΔF was pseudotyped with the homologous F protein (HaBacΔF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacΔF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacΔF-SeF virus was about ten times lower than that of HaBacΔF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F1 and F2 in the BVs of vHaBacΔF-HaF and vHaBacΔF-SeF, respectively, but the cleavage of SeF in vHaBacΔF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacΔF-SeF. Polyclonal antisera against HaF1 and SeF1 specifically neutralized the infection of vHaBacΔF-HaF and vHaBacΔF-SeF, respectively. HaF1 antiserum showed some cross-neutralization with vHaBacΔF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.
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- Plant
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Suppression of tobacco mosaic virus-induced hypersensitive-type necrotization in tobacco at high temperature is associated with downregulation of NADPH oxidase and superoxide and stimulation of dehydroascorbate reductase
More LessTissue necroses and resistance during the hypersensitive response (HR) of tobacco to tobacco mosaic virus (TMV) are overcome at temperatures above 28 °C and the virus multiplies to high levels in the originally resistant N-gene expressing plants. We have demonstrated that chemical compounds that generate reactive oxygen species (ROS) or directly applied hydrogen peroxide (H2O2) are able to induce HR-type necroses in TMV-inoculated Xanthi-nc tobacco even at high temperatures (e.g. 30 °C). The amount of superoxide (O2 •−) decreased, while H2O2 slightly increased in TMV- and mock-inoculated leaves at 30 °C, as compared with 20 °C. Activity of NADPH oxidase and mRNA levels of genes that encode NADPH oxidase and an alternative oxidase, respectively, were significantly lower, while activity of dehydroascorbate reductase was significantly higher at 30 °C, as compared with 20 °C. It was possible to reverse or suppress the chemically induced HR-type necrotization at 30 °C by the application of antioxidants, such as superoxide dismutase and catalase, demonstrating that the development of HR-type necroses indeed depends on a certain level of superoxide and other ROS. Importantly, high TMV levels at 30 °C were similar in infected plants, whether the HR-type necrotization developed or not. Suppression of virus multiplication in resistant, HR-producing tobacco at lower temperatures seems to be independent of the appearance of necroses but is associated with temperatures below 28 °C.
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Structure–function relationship between the tobamovirus TMV-Cg coat protein and the HR-like response
The tobamovirus TMV-Cg induces an HR-like response in Nicotiana tabacum cv. Xanthi nn sensitive plants lacking the N or N′ resistance genes. This response has been characterized by the appearance of necrotic lesions in the inoculated leaf and viral systemic spread, although the defence pathways are activated in the plant. A previous study demonstrated that the coat protein (CP) of TMV-Cg (CPCg) was the elicitor of this HR-like response. We examined the influence of four specific amino acid substitutions on the structure of CPCg, as well as on the development of the host response. To gain insights into the structural implications of these substitutions, a set of molecular dynamic experiments was performed using comparative models of wild-type and mutant CPCg as well as the CP of the U1 strain of TMV (CPU1), which is not recognized by the plants. A P21L mutation produces severe changes in the three-dimensional structure of CPCg and is more unstable when this subunit is laterally associated in silico. This result may explain the observed incapacity of this mutant to assemble virions. Two other CPCg mutations (R46G and S54K) overcome recognition by the plant and do not induce an HR-like response. A double CPCg mutant P21L-S54K recovered its capacity to form virions and to induce an HR-like response. Our results suggest that the structural integrity of the CP proteins is important for triggering the HR-like response.
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Supervirulent pseudorecombination and asymmetric synergism between genomic components of two distinct species of begomovirus associated with severe tomato leaf curl disease in India
More LessIsolates of two distinct begomovirus species, the severe strain of the species Tomato leaf curl New Delhi virus (tomato leaf curl New Delhi virus-[India:New Delhi:Severe:1992]; ToLCNDV-[IN:ND:Svr:92], bipartite) and the Varanasi strain of the species Tomato leaf curl Gujarat virus (tomato leaf curl Gujarat virus-[India:Varanasi:2001]; ToLCGV-[IN:Var:01], mono/bipartite) infect tomato (Lycopersicon esculentum) and cause severe yield losses in northern India. This study investigated the infectivity properties of genomic components of these two species. Both pseudorecombinants were infectious in Nicotiana benthamiana, Nicotiana tabacum and L. esculentum. Enhanced pathogenicity was observed when DNA-A of ToLCNDV-[IN:ND:Svr:92] was trans-complemented with ToLCGV-[IN:Var:01] DNA-B, and was consistently associated with an increase in accumulation of ToLCGV-[IN:Var:01] DNA-B. Mixed infection of ToLCNDV-[IN:ND:Svr:92] and ToLCGV-[IN:Var:01] always showed extremely severe symptoms, suggesting a synergistic interaction between these two viruses. Southern blot analysis of viral DNAs from infected plants showed a significantly higher level of accumulation of both ToLCNDV components and DNA-B of ToLCGV-[IN:Var:01] with no alteration to levels of DNA-A of ToLCGV-[IN:Var:01]. Symptom development and/or higher infectivity of the supervirulent pseudorecombinants correlated with the increased levels of DNA-B accumulation. Protoplast replication assays revealed that enhanced infectivity by the pseudorecombinant occurred at the level of replication, as DNA-A of ToLCNDV-[IN:ND:Svr:92] enhanced ToLCGV-[IN:Var:01] DNA-B replication, whose accumulation was in turn increased by ToLCGV-[IN:Var:01] DNA-A. This is the first report demonstrating a virulent pseudorecombinant between two distinct species of begomoviruses that infect tomato, and is the second report on synergism between begomoviruses. The results revealed that ToLCGV-[IN:Var:01] DNA-B is capable of associating with different DNA-A components, despite having different iteron sequences.
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Cylindrical inclusion protein of potato virus A is associated with a subpopulation of particles isolated from infected plants
Potato virus A (PVA) particles were purified by centrifugation through a 30 % sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5–40 % sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. CI was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of CI. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the CI present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for CI-mediated functions.
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