- Volume 87, Issue 9, 2006
Volume 87, Issue 9, 2006
- Animal
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- RNA viruses
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L-SIGN (CD209L) isoforms differently mediate trans-infection of hepatoma cells by hepatitis C virus pseudoparticles
L-SIGN is a C-type lectin that is expressed on liver sinusoidal endothelial cells. Capture of Hepatitis C virus (HCV) by this receptor results in trans-infection of hepatoma cells. L-SIGN alleles have been identified that encode between three and nine tandem repeats of a 23 residue stretch in the juxtamembrane oligomerization domain. Here, it was shown that these repeat-region isoforms are expressed at the surface of mammalian cells and variably bind HCV envelope glycoprotein E2 and HCV pseudoparticles. Differences in binding were reflected in trans-infection efficiency, which was highest for isoform 7 and lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphism could influence the establishment and progression of HCV infection.
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High-density lipoproteins reduce the neutralizing effect of hepatitis C virus (HCV)-infected patient antibodies by promoting HCV entry
The neutralizing activity of anti-hepatitis C virus (HCV) antibodies is attenuated by a factor present in human sera, which has been proposed to be high-density lipoproteins (HDLs). HDLs have also been shown to facilitate the entry of HCV pseudoparticles (HCVpp) into target cells. Here, the aim of the study was to determine whether HDL-mediated facilitation of HCVpp and infectious HCV (HCVcc) entry and attenuation of neutralization are two related phenomena. The data indicated that HDLs attenuate neutralization at a constant rate. In addition, as for HDL-mediated facilitation of HCVpp entry, attenuation of neutralization depended on the expression of the scavenger receptor BI (SR-BI) and its selective lipid-uptake function. Finally, kinetic experiments showed that HDL-mediated facilitation of HCVpp entry is more rapid than virus neutralization. Altogether, these observations indicate that HCV is exploiting the physiological activity of SR-BI for promoting its entry into target cells, which consequently also protects the virus against neutralizing antibodies.
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Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Studies of the early steps of HCV infection have been hampered by the lack of convenient in vitro or in vivo models. Although several cell-surface molecules that mediate the binding of HCV envelope proteins to target cells have been identified, mechanisms of viral entry into human hepatocytes are still poorly understood. Vesicular stomatitis virus/HCV pseudotyped viruses expressing the HCV envelope glycoproteins on the viral envelope were generated and it was found that their entry into human hepatocytes required co-expression of E1 and E2 on the pseudotype surface. Neutralization of pseudotype infection by anti-HCV antibodies suggested that cellular entry was mediated by HCV envelope glycoproteins and by previously characterized cell-surface molecules, including CD81. An entry assay based on the release of a fluorochrome from labelled HCV pseudotypes provided evidence for a pH-dependent fusion of the pseudotype envelope with a cellular compartment. By using a panel of endocytosis inhibitors, it is postulated that penetration of HCV into primary cultures of hepatocytes takes place by clathrin-mediated endocytosis.
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Comparative analysis reveals no consistent association between the secondary structure of the 3′-untranslated region of dengue viruses and disease syndrome
A comparative analysis was performed of the 3′-untranslated region (UTR) of Dengue virus (DENV) sampled from Bangkok, Thailand, over a 30 year period and representing all four serotypes. Considerable genetic variation was observed both within and among serotypes. Notably, a full-length version of the critical 3′-long stable hairpin structure was absent from some isolates, suggesting the occurrence of complex structural interactions within the 3′-UTR, including the influence of upstream mutations. The Thai sequences were then combined with 61 globally sampled isolates of DENV taken from patients with either dengue fever or severe dengue disease. No consistent association was found between 3′-UTR secondary structure and the clinical outcome of DENV infection, although some evidence for a trend in this direction was observed in DENV-2. It was concluded that the 3′-UTR is not the sole determinant of DENV virulence in nature, although variation in secondary structure may greatly influence viral fitness.
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Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA
More LessDengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3.
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The 3′ untranslated regions of Kamiti River virus and Cell fusing agent virus originated by self-duplication
More LessPreviously, it was shown that the 3′ untranslated region (3′UTR) of Kamiti River virus (KRV) is nearly twice as long as the 3′UTR of other flaviviruses (1208 nucleotides compared with 730 nucleotides for the longest 3′UTR of any virus in the Tick-borne encephalitis virus species). Additionally, KRV and the closely related Cell fusing agent virus (CFAV) were shown to contain two short, almost perfect repeat sequences of 67 nucleotides. However, the construction of a robust comparative nucleotide alignment has now revealed that the double-length 3′UTR and the direct repeats resulted from the virtually complete duplication of a primordial KRV 3′UTR. We also propose that the CFAV 3′UTR was derived from a KRV-like precursor sequence with a large deletion that nevertheless preserved the two direct repeat sequences. These data provide new insights into the evolution of the flavivirus 3′UTR.
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Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis
More LessNorovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.
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Proteome alterations in human host cells infected with coxsackievirus B3
Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. Despite that, host protein responses, which characterize CVB3-induced diseases, have not yet been determined in detail. To investigate the nature of modified protein patterns in infected human cells compared with uninfected cells, two-dimensional gel electrophoresis in combination with matrix-assisted laser desorption/ionization-mass spectrometry were used. The regulated proteins, e.g. nucleophosmin (nucleolar protein B23), lamin, the RNA-binding protein UNR and the p38 mitogen-activated protein kinase, were sorted according to their functional groups and interpreted in the context of the myocarditis process.
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Influenza A virus NS1 protein does not suppress RNA interference in mammalian cells
More LessInfluenza A virus NS1 protein has been shown to suppress RNA interference (RNAi) in plants and Drosophila. Although it has not been demonstrated experimentally, NS1 has also been thought to inhibit RNAi in mammals as well as being an antagonist of interferon. In this study, the influence of NS1 protein from influenza virus strain A/WSN/33 on RNAi in mammalian cells was investigated. While transiently or stably expressed NS1 was fully competent to inhibit the interferon pathway in cultured cells, it did not suppress RNAi-mediated silencing of different reporter genes. These findings imply a significant difference in RNAi mechanism between mammals and plants.
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In vivo correlates of infectious salmon anemia virus pathogenesis in fish
More LessThe phenotypic correlates of pathogenicity for Infectious salmon anemia virus (ISAV) in salmonid fishes have not been thoroughly studied to date. In this study, a comparison was made of 13 different strains of ISAV, isolated from different geographical regions between 1997 and 2004, for their infectivity in three fish species [Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss)]. When the different virus isolates were used at an approximate inoculum dose of 106 TCID50 in 0.2 ml per fish, it was found that the most virulent strains had an acute mortality phase in Atlantic salmon that started at 10–13 days post-inoculation and lasted for 9–15 days with a cumulative mortality of ⩾90 %. These highly pathogenic strains also caused low mortality in rainbow trout, albeit later in infection. Viruses with a more delayed or protracted mortality phase resulting in cumulative mortalities of 50–89 % in Atlantic salmon were considered to be of intermediate pathogenicity and isolates with cumulative mortalities of ⩽49 % were considered to be of low pathogenicity. On this basis, three of the ISAV isolates showed a high-, eight an intermediate- and two a low-pathogenicity phenotype in Atlantic salmon. Coho salmon were resistant to all ISAV isolates. These results confirmed that there is variation in pathogenicity among ISAV strains for Atlantic salmon and rainbow trout, and that other salmonid species such as coho salmon can carry highly pathogenic strains of ISAV without showing signs of disease. The identified pathogenicity phenotypes may aid in the identification of molecular markers of ISAV virulence.
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Inhibition of vesicular stomatitis virus infection in epithelial cells by alpha interferon-induced soluble secreted proteins
More LessInterferons (IFNs) are potent antiviral cytokines that inhibit infection by a wide spectrum of viruses by activating the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Several IFN-induced antiviral proteins including 2′,5′-oligoadenylate synthetase, dsRNA-activated protein kinase and Mx play a critical role in conferring the antiviral properties of IFN. However, studies have shown that additional antiviral factors are involved in addition to these proteins during IFN-mediated antiviral action. In an effort to characterize these novel antiviral factors, the antiviral mechanism of alpha IFN (IFN-α) against vesicular stomatitis virus (VSV) was investigated in human lung epithelial A549 cells. These studies demonstrated that soluble secreted antiviral proteins as the constituents of conditioned medium prepared from IFN-α-treated cells reduced VSV infectivity by more than 2 logs, compared with a 4 log inhibition observed following treatment of cells with IFN-α. The antiviral mechanism of these secreted proteins appeared to act at the level of cellular entry of VSV. Interestingly, the IFN-α-induced antiviral proteins were secreted independently of STAT1 (an essential component of the JAK/STAT pathway), demonstrating that the release of such extracellular soluble antiviral proteins from cells may represent an alternative mechanism of the antiviral defence strategy of IFN towards VSV infection.
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Lyssavirus infection activates interferon gene expression in the brain
To investigate the innate immune response within the brain to lyssavirus infection, key transcripts indicative of innate defences were measured in a mouse model system. Following infection with Rabies virus, transcript levels for type 1 interferons (IFN-α and -β), the inflammatory mediator interleukin 6 (IL-6) and the antiviral protein Mx1 increased in the brains of mice. Intracranial inoculation resulted in the early detection of virus replication and rapid expression within the brain of the innate immune response genes. Transcripts for type 1 IFNs declined as the disease progressed. Peripheral, extraneural inoculation delayed the host response until virus entered the brain, but then resulted in a large increase in the level of IFN-β, IL-6 and Mx1 transcripts. Induction of this response was also observed following infection with the related European bat lyssaviruses, a group of zoonotic viruses capable of causing fatal, rabies-like disease in mammalian species.
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Gag–Pol bearing a reverse transcriptase drug-resistant mutation influences viral genomic RNA incorporation into human immunodeficiency virus type 1 particles
More LessThe unspliced human immunodeficiency virus type 1 (HIV-1) RNA is both the messenger for Gag and Gag–Pol and the viral genomic RNA (vRNA) that is packaged into the virion. Although Gag alone is sufficient for the incorporation of vRNA into virus particles, Gag–Pol molecules play an important role in vRNA dimerization and virion maturation. Here, a cis model for vRNA packaging was demonstrated, in which nascent Gag–Pol molecules were preferentially co-encapsulated with their cognate RNA used as the template. Genome-incorporation frequencies were evaluated for two distinct HIV-1 proviral clones differing in their ability to respond to nevirapine (NVP) treatment in one round of infection. It was shown that, under NVP selection, there was a twofold-higher incorporation of vRNAs and integration of provirus genome carrying NVP resistance when compared with the wild-type counterpart. Although cis incorporation has been already demonstrated for Gag, the novelty of these findings is that newly acquired resistant mutations in Gag–Pol will select their specific genomic RNA during virus replication, thus rapidly increasing the chance of the emergence of resistant viruses during the course of anti-retroviral treatment.
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Transmission of Moloney murine leukemia virus (ts-1) by breast milk
More LessA murine model has been developed to study maternal transmission of the temperature-sensitive Moloney murine leukemia virus (ts-1). The goal of this study was to confirm early and late mother-to-offspring transmission of the virus and demonstrate transmission via breast milk. A series of six experiments was performed using six groups of BALB/c mice. Group 1 consisted of pups born to ts-1-infected mothers removed at birth to suckle from surrogate uninfected mothers. Groups 2 and 5 consisted of pups born to ts-1-infected mothers that suckled from ts-1-infected mothers (surrogate and biological). Group 3 consisted of non-infected pups removed at birth to suckle from ts-1-infected mothers. Groups 4 and 6 consisted of non-infected pups suckled from non-infected mothers. The combined in utero, intrapartum and breast-milk infection rate was 100 % to the offspring (groups 2 and 5). The in utero to early post-partum group (group 1) had an infection rate of 78 %. Breast milk alone (group 3) resulted in a 97 % infection rate. Control groups (groups 4 and 6) had a 0 % infection rate. The relative frequency of maternal CD4+ cells in peripheral blood mononuclear cells was consistently lower in infected mothers, whilst offspring did not show a significant decrease in CD4+ frequency. Pups infected via breast milk had a lower CD4+ frequency (group 3) than those infected by the uterine and/or intrapartum route (group 1). Breast milk from ts-1-infected mothers appears to be highly infectious for neonatal BALB/c mice.
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E (XSR) element contributes to the oncogenicity of Avian leukosis virus (subgroup J)
More LessAmong the six subgroups of Avian leukosis virus (ALV) that infect chickens, subgroup J (ALV-J) was isolated from meat-type chickens where it predominantly induces myeloid leukosis (ML) and erythroblastosis (EB). The sequence of HPRS-103, the ALV-J prototype virus, shows several distinct features, one of which is the presence of a distinct hairpin stem–loop structure called the E (also called XSR) element in the 3′ untranslated region. In order to determine the role of the E element in ALV-induced pathogenicity, a comparison was made of the oncogenicity of viruses derived from the provirus clones of parental and E element-deleted HPRS-103 viruses in two genetically distinct lines of birds. In line 15I birds, deletion of the E element had profound effects on virus replication in vivo, as only 55 % of birds showed evidence of infection, compared with 100 % infection by the parental virus. Furthermore, none of the line 15I birds infected with this virus developed tumours, indicating that the E element does contribute to the oncogenicity of the virus. On the other hand, deletion of the E element had only a marginal effect on the incidence of tumours in line 0 birds. These results indicate that, although the E element per se is not absolutely essential for tumour induction by this subgroup of viruses, it does contribute to oncogenicity in certain genetic lines of chicken.
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- DNA viruses
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Generation of an adenoviral vaccine vector based on simian adenovirus 21
Adenoviral vectors can be used to generate potent humoral and cellular immune responses to transgene products. Use of adenoviral vectors based on non-human isolates may allow for their utilization in populations harbouring neutralizing antibodies to common human serotypes. A vector chimera was constructed using simian adenovirus 22 (a serotype belonging to the species Human adenovirus E) and simian adenovirus 21 (a serotype belonging to the species Human adenovirus B) expressing the Ebola (Zaire) virus glycoprotein (Ad C5/C1-ZGP). This chimeric adenovirus vector was used as a model to test its efficacy as a genetic vaccine and comparisons were made to a vector based on the commonly used human adenovirus C serotype 5 (Adhu5-ZGP). Ebola glycoprotein-specific T- and B-cell responses were measured in B10BR mice vaccinated with either Adhu5-ZGP or Ad C5/C1-ZGP vectors. Both vectors resulted in Ebola glycoprotein-specific gamma interferon-expressing T cells, although the Ad C5/C1-ZGP vector appeared to induce lower frequencies with kinetics slower than those elicited by the Adhu5-ZGP vector. The total immunoglobulin G response to Ebola glycoprotein was similar in sera from mice vaccinated with either vector. Two rhesus macaques vaccinated with the Ad C5/C1-ZGP vector were found to mount T-cell and antibody responses to the Ebola glycoprotein. It was found that a single administration of the chimeric Ad C5/C1-ZGP vector protected mice against a lethal challenge with a mouse-adapted strain of the Ebola (Zaire) virus.
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Role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting
More LessLiver tropism hampers systemic administration of adenovirus in gene therapy and virotherapy. In consequence, tumour targeting requires the combination of capsid modifications that abrogate liver transduction and redirect adenoviral vectors to tumour cells. Coxsackievirus and adenovirus receptor (CAR), integrins and heparan sulfate glycosaminoglycans (HSG) are receptors involved in adenovirus type 5 (Ad5) entry into cells. The in vitro and in vivo properties of Ad5 vectors unable to bind CAR, integrins and HSG with and without Arg–Gly–Asp (RGD) inserted at the HI loop of the fiber were studied. As was previously observed with CAR-ablated vectors, CAR and integrin double binding-ablated vectors transduced hepatocytes less efficiently in vitro but not in vivo. On the contrary, the role of HSG on Ad5 infectivity was evident in vitro only when CAR binding was abrogated, but the shaft mutation that ablated HSG binding on the background of a normal capsid was sufficient to abrogate liver transduction in vivo. The insertion of amino acids RGD at the HI loop in a shaft-mutated fiber only partially rescued integrin-mediated infectivity. These results indicate that the shaft mutation precluded HSG binding and affected the structure of the fiber. The insertion of ligands at the hexon or protein IX may be required to benefit from the fiber shaft mutation-detargeting properties.
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Analysis of the interaction between RGD-expressing adenovirus type 5 fiber knob domains and αvβ3 integrin reveals distinct binding profiles and intracellular trafficking
More LessAdenovirus (Ad) vectors are used widely for experimental and therapeutic gene transfer. Ad-mediated gene delivery is often inefficient and, thus, there is considerable interest in developing Ad vectors that overcome biological barriers to efficient virus uptake. For this strategy to succeed, it is imperative that the interaction between such Ad vectors and their novel receptors is well understood. In this study, three surface-exposed loops (HI, CD and IJ loops) on the Ad5 fiber knob domain were selected as sites for insertion of an αvβ3 integrin-binding RGD sequence. Three RGD-containing Ad5 fiber knob-domain mutants were produced as recombinant proteins and all were shown to interact with soluble αvβ3 integrin by using biomolecular cell-free assays. Cell adsorption and subsequent internalization and intracellular trafficking of each of these proteins were assessed by confocal microscopy. Whilst the Ad5 fiber knob domain expressing the RGD sequence in the HI and CD loops bound with similar association and dissociation profiles, the fiber knob domain expressing the RGD sequence in the IJ loop bound with slower association and faster dissociation rates. By using molecular modelling, it was shown that the Ad5 fiber knob domain in which the RGD peptide was expressed in the IJ loop was only capable of binding to one αvβ3 integrin molecule per trimer. In contrast, fiber knob domains in which the RGD peptide was expressed in the HI and CD loops were capable of binding to one integrin molecule per monomer. These differences in the interactions between each mutant and αvβ3 may explain our observation that the three RGD-bearing Ad5 fiber knob domains demonstrated similar internalization rates, but distinct patterns of endosomal transport and escape.
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A molecular approach to the identification of cytotoxic T-lymphocyte epitopes within equine herpesvirus 1
Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.
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The UL4 gene of pseudorabies virus encodes a minor infected-cell protein that is dispensable for virus replication
More LessAlthough homologues of the open reading frame (ORF) UL4 of herpes simplex virus 1 (Human herpesvirus 1) have been found in the genomes of all hitherto-analysed alphaherpesviruses, little is known about their function. In a project to analyse systematically, in an isogenic and standardized assay system, the gene products of the alphaherpesvirus pseudorabies virus (PrV; Suid herpesvirus 1), the PrV UL4 gene product was identified using a monospecific rabbit antiserum prepared against a bacterial fusion protein. Western blot and immunofluorescence analyses revealed that the 146 codon UL4 ORF of PrV was translated into a nuclear 15 kDa protein which was detectable from 6 h after infection of rabbit kidney cells, but was not found in purified virus particles. For functional analysis, a UL4-negative virus recombinant (PrV-ΔUL4F) was generated by mutagenesis of an infectious full-length clone of the PrV genome in E. coli. PrV-ΔUL4F was replication-competent in rabbit kidney cells, and plaque formation was not affected by the mutation. However, maximum virus titres of PrV-ΔUL4F were decreased about fivefold compared with wild-type PrV, and electron microscopy of infected cells demonstrated an impairment of release of mature virions. This growth defect of PrV-ΔUL4F could be corrected completely by propagation in UL4-expressing cells. Correlating with the inconspicuous in vitro phenotype, neurovirulence of PrV-ΔUL4F was also not affected significantly. Thus, UL4 encodes a non-structural protein of PrV that enhances virion formation but is not essential for PrV replication in vitro or in vivo.
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Evidence of ancient papillomavirus recombination
An open question amongst papillomavirus taxonomists is whether recombination has featured in the evolutionary history of these viruses. Since the onset of the global AIDS epidemic, the question is somewhat less academic, because immune-compromised human immunodeficiency virus patients are often co-infected with extraordinarily diverse mixtures of human papillomavirus (HPV) types. It is expected that these conditions may facilitate the emergence of HPV recombinants, some of which might have novel pathogenic properties. Here, a range of rigorous analyses is applied to full-genome sequences of papillomaviruses to provide convincing statistical and phylogenetic evidence that evolutionarily relevant papillomavirus recombination can occur.
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A granule cell neuron-associated JC virus variant has a unique deletion in the VP1 gene
More LessThe human polyomavirus JC (JCV) typically infects glial cells and is the aetiological agent of progressive multifocal leukoencephalopathy (PML), which occurs in immunosuppressed individuals. The full-length sequence of a granule cell neuron-tropic JCV variant, JCVGCN1, associated with lytic infection of granule cell neurons and cerebellar atrophy in a human immunodeficiency virus-infected patient with PML was determined and compared with the sequence of the JCV isolate from the classic PML lesions present in the hemispheric white matter of the same individual (JCVHWM). A unique deletion was found in the C terminus of the VP1 gene of JCVGCN1, which encodes the major capsid protein, resulting in a frame shift and a total change of the C-terminal amino acid sequence of this protein. This deletion was not present in JCVHWM, suggesting that this mutation may be instrumental in facilitating entry or replication of JCV into granule cell neurons.
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Cell death in bovine parvovirus-infected embryonic bovine tracheal cells is mediated by necrosis rather than apoptosis
The helper-independent bovine parvovirus (BPV) was studied to determine its effect on host embryonic bovine tracheal (EBTr) cells: whether the ultimate outcome of infection results in apoptotic cell death or cell death by necrosis. Infected cells were observed for changes marking apoptosis. Observations of alterations in nuclear morphology, membrane changes, apoptotic body formation, membrane phosphatidylserine inversions, caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. On the other hand, at the end of the virus replication cycle, infected cells released viral haemagglutinin and infectious virus particles, as would be expected from cell membrane failure. Moreover, the infected cells released lactate dehydrogenase (LDH), release of which is a marker of necrosis. LDH release into the cell medium correlated directly with viral m.o.i. and time post-infection. Furthermore, assessment of mitochondrial dehydrogenase activity was consistent with cell death by necrosis. Taken together, these findings indicate that cell death in BPV-infected EBTr cells is due to necrosis, as defined by infected-cell membrane failure and release of the cell contents into the extracellular environment.
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Gene organization and complete sequence of the Hyphantria cunea nucleopolyhedrovirus genome
More LessThe whole-genome sequence of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) was analysed. The entire nucleotide sequence of the HycuNPV genome was 132 959 bp long, with a G+C content of 45.1 mol%. A total of 148 open reading frames (ORFs) consisting of more than 50 aa were encoded by the genome. HycuNPV shares more than 122 ORFs with other lepidopteran group I NPVs, including Autographa californica MNPV, Bombyx mori NPV, Choristoneura fumiferana MNPV (CfMNPV), Choristoneura fumiferana defective NPV, Epiphyas postvittana MNPV and Orgyia pseudotsugata MNPV (OpMNPV). Six ORFs are identified as being unique to HycuNPV. Most of the HycuNPV ORFs showed higher similarity to CfMNPV and OpMNPV ORFs than to those of the other group I NPVs. HycuNPV encodes two conotoxin-like homologues (ctls), which are observed only in OpMNPV in group I NPVs. HycuNPV encodes three inhibitors of apoptosis (iaps), hycu-iap-1, hycu-iap-2 and hycu-iap-3, a feature that it shares only with CfMNPV. In addition, six homologous regions (hrs) are identified in the HycuNPV genome. These hrs are located in regions similar to those of the OpMNPV hrs, but different from most of the CfMNPV hrs. Based on the close phylogenetic relationship and conservation of group I NPV-specific genes, such as gp64, ie-2 and ptp-1, it is concluded that HycuNPV belongs to the group I NPVs and is most similar to CfMNPV or OpMNPV.
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Open reading frame 132 of Heliocoverpa armigera nucleopolyhedrovirus encodes a functional per os infectivity factor (PIF-2)
Open reading frame 132 (Ha132) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is a homologue of per os infectivity factor 2 (pif-2) of Spodoptera exigua multiple nucleopolyhedrovirus. Sequence analysis indicated that Ha132 encoded a protein of 383 aa with a predicted molecular mass of 44.5 kDa. Alignment of HA132 and its baculovirus homologues revealed that HA132 was highly conserved among baculoviruses, with 14 absolutely conserved cysteine residues. RT-PCR indicated that Ha132 was first transcribed at 24 h post-infection. Western blot analysis showed that a 43 kDa band was detectable in HearNPV-infected HzAM1 cells from 36 h post-infection. Western blots also indicated that HA132 was a component of the occlusion-derived virus, but not of budded virus. Deletion of Ha132 from HearNPV abolished per os infectivity, but had no effect on the infectivity of the budded virus phenotype.
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- Plant Viruses
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An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo
More LessIt was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)CP,148 CR upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem–loop (SL) region of CP subgenomic promoters (TMV U1-CP–GFP/SL-mut and crTMV-CP–GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP–GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP–GFP/SL-mut, whereas a small amount of CP–GFP synthesis was obtained in crTMV-CP–GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.
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Domains of tobacco mosaic virus movement protein essential for its membrane association
More LessA series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains.
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Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions
The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.
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Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type
The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5′ regions of the two genomic RNAs contain a ‘cap’ structure and poly(A) tails were identified in the 3′-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.
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Potato virus X RNA-mediated assembly of single-tailed ternary ‘coat protein–RNA–movement protein’ complexes
Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5′-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP–RNA interactions resulting in head formation prevailed over TGBp1–RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5′ end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP–RNA STPs are produced, due to encapsidation of the 5′-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.
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Random mutagenesis of wheat streak mosaic virus HC-Pro: non-infectious interfering mutations in a gene dispensable for systemic infection of plants
More LessMutations within the HC-Pro coding region of Wheat streak mosaic virus (WSMV) were introduced by misincorporation during PCR and evaluated for phenotype within the context of an infectious clone. Nine synonymous substitutions and 15 of 25 non-synonymous substitutions had no phenotypic effect. Four non-synonymous substitutions, including one that reverted consistently to wild type, resulted in attenuated systemic infection. Six non-synonymous substitutions and one nonsense substitution abolished systemic infectivity. Mutants bearing the GUS reporter gene were evaluated for the ability to establish primary infection foci. All attenuated mutants and two systemic infection-deficient mutants produced localized regions of GUS expression on inoculated leaves 3 days post-inoculation. In vitro assays revealed that mutants able to establish infection foci retained HC-Pro proteinase activity. Among mutants unable to establish infection foci, HC-Pro proteinase activity was retained, reduced or absent. As a complete HC-Pro deletion mutant can infect plants systemically, certain substitutions in this dispensable gene probably prevented infection of WSMV via interference.
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In vitro-reassembled plant virus-like particles for loading of polyacids
More LessThe coat protein (CP) of certain plant viruses may reassemble into empty virus-like particles (VLPs) and these protein cages may serve as potential drug delivery platforms. In this paper, the production of novel VLPs from the Hibiscus chlorotic ringspot virus (HCRSV) is reported and the capacity to load foreign materials was characterized. VLPs were readily produced by destabilizing the HCRSV in 8 M urea or Tris buffer pH 8, in the absence of calcium ions, followed by removal of viral RNA by ultrahigh-speed centrifugation and the reassembly of the CP in sodium acetate buffer pH 5. The loading of foreign materials into the VLPs was dependent on electrostatic interactions. Anionic polyacids, such as polystyrenesulfonic acid and polyacrylic acid, were successfully loaded but neutrally charged dextran molecules were not. The molecular-mass threshold for the polyacid cargo was about 13 kDa, due to the poor retention of smaller molecules, which readily diffused through the holes between the S domains present on the surface of the VLPs. These holes precluded the entry of large molecules, but allowed smaller molecules to enter or exit. The polyacid-loaded VLPs had comparable size, morphology and surface-charge density to the native HCRSV, and the amount of polyacids loaded was comparable to the weight of the native genomic materials. The conditions applied to disassembly–reassembly of the virions did not change the structural conformation of the CP. HCRSV-derived VLPs may provide a promising nano-sized protein cage for delivery of anionic drug molecules.
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Role of human cytomegalovirus UL131A in cell type-specific virus entry and release
The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.
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Coronaviruses in bent-winged bats (Miniopterus spp.)
More LessA novel group 1 coronavirus was previously identified in bent-winged bats (Miniopterus spp.). Here, results are described from our ongoing surveillance of these bats for coronaviruses. These findings show that group 1 coronaviruses are endemic in these bat populations in Hong Kong. Genetic analysis of these viruses indicates that there are at least four different, but closely related, group 1 coronaviruses (bat-CoV 1A, 1B, HKU7 and HKU8) circulating in bent-winged bats. Phylogenetic analysis revealed that these group 1 bat coronaviruses have descended from a common ancestor and that these viruses have been established in these bats for a long period of time. These data provide a better understanding of the emergence and evolution of coronaviruses. Bat-CoV 1A and 1B were detected in apparently healthy Miniopterus magnater and Miniopterus pusillus, respectively, on repeated sampling occasions at a single habitat, suggesting that these viruses have established a persistent infection in these populations.
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A humanized murine monoclonal antibody protects mice either before or after challenge with virulent Venezuelan equine encephalomyelitis virus
More LessA humanized monoclonal antibody (mAb) has been developed and its potential to protect from or cure a Venezuelan equine encephalomyelitis virus (VEEV) infection was evaluated. The VEEV-neutralizing, protective murine mAb 3B4C-4 was humanized using combinatorial antibody libraries and phage-display technology. Humanized VEEV-binding Fabs were evaluated for virus-neutralizing capacity, then selected Fabs were converted to whole immunoglobulin (Ig) G1, and stable cell lines were generated. The humanized mAb Hy4-26C, designated Hy4 IgG, had virus-neutralizing capacity similar to that of 3B4C-4. Passive antibody protection studies with purified Hy4 IgG were performed in adult Swiss Webster mice. As little as 100 ng Hy4 IgG protected 90 % of mice challenged with 100 intraperitoneal (i.p.) mean morbidity (MD50) doses of virulent VEEV (Trinidad donkey) 24 h after antibody transfer; also, 500 μg Hy4 IgG protected 80 % of mice inoculated with 100 intranasal MD50 doses of VEEV. Moreover, 10 μg passive Hy4 IgG protected 70 % of mice from a VEEV challenge dose as great as 107 i.p. MD50. Hy4 IgG also protected mice from challenge with another epizootic VEEV variety, 1C (P676). Importantly, therapeutic administration of the humanized mAb to mice already infected with VEEV cured 90 % of mice treated with Hy4 IgG within 1 h of VEEV inoculation and 75 % of mice treated 24 h after virus infection.
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Volumes and issues
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Volume 105 (2024)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)