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Volume 87,
Issue 4,
2006
Volume 87, Issue 4, 2006
- Animal
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- RNA viruses
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Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly
Hepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly.
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Antiviral effect of α-glucosidase inhibitors on viral morphogenesis and binding properties of hepatitis C virus-like particles
Hepatitis C virus (HCV) infections are a major public-health concern. New antiviral drugs are needed urgently to complement and improve the efficacy of current chemotherapies. The morphogenesis of HCV represents an interesting, and still unexploited, novel molecular target. α-Glucosidase inhibitors derived from the glucose analogue deoxynojirimycin (DNJ) inhibit viral morphogenesis in cellulo via perturbation of the N-glycosylation pathway and hence the misfolding of viral glycoproteins that depend on certain N-glycans for correct folding. Due to the heavy N-glycosylation of HCV glycoproteins, it was hypothesized that such inhibitors would also affect HCV morphogenesis. To study the effect of α-glucosidase inhibitors on viral morphogenesis and binding properties, HCV virus-like particles (VLPs) were produced by using baculovirus loaded with HCV structural-protein genes. Here, it is demonstrated that, in the presence of these α-glucosidase inhibitors, viral glycoproteins synthesized and retained in the endoplasmic reticulum (i) contain unprocessed, triglucosylated N-glycans, (ii) are impaired in their interaction with calnexin and (iii) are at least partially misfolded. Moreover, it is shown that, although the production of VLPs is not affected by α-glucosidase inhibitors, these VLPs contain unprocessed, triglucosylated N-glycans and potentially misfolded glycoproteins. Finally, it is demonstrated that VLPs produced in the presence of α-glucosidase inhibitors have impaired binding properties to hepatoma cells. The inhibitors of morphogenesis studied here target steps of the HCV viral cycle that may prevent or delay viral resistance. These α-glucosidase inhibitors may prove to be useful molecules to fight HCV infection in combination protocols.
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Structure and age of genetic diversity of dengue virus type 2 in Thailand
Dengue virus type 2 (DENV-2) is a common viral infection and an important health concern in South-East Asia. To determine the molecular evolution of DENV-2 in Thailand, 105 isolates of the E (envelope) gene and 10 complete genomes sampled over a 27 year period were sequenced. Phylogenetic analysis of these data revealed that three genotypes of DENV-2 have circulated in Thailand, although, since 1991, only viruses assigned to Asian genotype I have been sampled from the population. A broader analysis of 35 complete genomes of DENV-2 revealed that most amino acids are subject to strong selective constraints, indicative of widespread purifying selection against deleterious mutations. This was further supported by an analysis of genome-wide substitution rates, which indicated that DENV-2 fixes approximately 10 mutations per genome per year, far lower than expected from its mutational dynamics. Finally, estimates of the age of DENV-2 were remarkably consistent among genes, indicating that the current genetic diversity in this virus probably arose within the last 120 years, concordant with the first determination of the aetiology of dengue disease.
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Molecular evolution of dengue 2 virus in Puerto Rico: positive selection in the viral envelope accompanies clade reintroduction
Dengue virus is a circumtropical, mosquito-borne flavivirus that infects 50–100 million people each year and is expanding in both range and prevalence. Of the four co-circulating viral serotypes (DENV-1 to DENV-4) that cause mild to severe febrile disease, DENV-2 has been implicated in the onset of dengue haemorrhagic fever (DHF) in the Americas in the early 1980s. To identify patterns of genetic change since DENV-2's reintroduction into the region, molecular evolution in DENV-2 from Puerto Rico (PR) and surrounding countries was examined over a 20 year period of fluctuating disease incidence. Structural genes (over 20 % of the viral genome), which affect viral packaging, host-cell entry and immune response, were sequenced for 91 DENV-2 isolates derived from both low- and high-prevalence years. Phylogenetic analyses indicated that DENV-2 outbreaks in PR have been caused by viruses assigned to subtype IIIb, originally from Asia. Variation amongst DENV-2 viruses in PR has since largely arisen in situ, except for a lineage-replacement event in 1994 that appears to have non-PR New World origins. Although most structural genes have remained relatively conserved since the 1980s, strong evidence was found for positive selection acting on a number of amino acid sites in the envelope gene, which have also been important in defining phylogenetic structure. Some of these changes are exhibited by the multiple lineages present in 1994, during the largest Puerto Rican outbreak of dengue, suggesting that they may have altered disease dynamics, although their functional significance will require further investigation.
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Genome analysis and phylogenetic relationships between east, central and west African isolates of Yellow fever virus
Yellow fever virus (YFV), a reemerging disease agent in Africa and South America, is the prototype member of the genus Flavivirus. Based on examination of the prM/M, E and 3′ non-coding regions of the YFV genome, previous studies have identified seven genotypes of YFV, including the Angolan, east/central African and east African genotypes, which are highly divergent from the prototype strain Asibi. In this study, full genome analysis was used to expand upon these genetic relationships as well as on the very limited full genome database for YFV. This study was the first to investigate genomic sequences of YFV strains from east and central Africa (Angola71, Uganda48a and Ethiopia61b). All three viruses had genomes of 10 823 nt in length. Compared with the prototype strain Asibi (from west Africa) they were approximately 25 % divergent in nucleotide sequence and 7 % divergent in amino acid sequence. Comparison of multiple flaviviruses in the N-terminal region of NS4B showed that amino acid sequences were variable and that west African strains of YFV had an amino acid deletion at residue 21. Additionally, N-linked glycosylation sites were conserved between viral genotypes, while codon usage varied between strains.
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Genetic and antigenic diversity among noroviruses
Grant S. Hansman, Katsuro Natori, Haruko Shirato-Horikoshi, Satoko Ogawa, Tomoichiro Oka, Kazuhiko Katayama, Tomoyuki Tanaka, Tatsuya Miyoshi, Kenji Sakae, Shinichi Kobayashi, Michiyo Shinohara, Kazue Uchida, Nakao Sakurai, Kuniko Shinozaki, Mineyuki Okada, Yoshiyuki Seto, Kunio Kamata, Noriyo Nagata, Keiko Tanaka, Tatsuo Miyamura and Naokazu TakedaHuman norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1–14 and GII/1–17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.
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Recombination of Feline calicivirus within an endemically infected cat colony
More LessTo understand the evolution of the family Caliciviridae, the persistence of Feline calicivirus (FCV) was studied within an endemically infected cat colony. Polymerase and capsid sequences were analysed for 34 FCV isolates obtained over a 4 year period. Initially, the colony was infected with one strain of virus, but a second distinct strain was later identified. Subsequently, the emergence of a recombinant virus was observed, containing elements of both of the strains circulating within the colony. The recombination event mapped close to the ORF1/ORF2 junction. This is consistent with recombination in other caliciviruses, suggesting a common mechanism within this family. This is the first report of recombination within the genus Vesivirus in the family Caliciviridae and the first time that a recombination event has been observed where the parental strains have also been identified.
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Hepatitis C virus-related internal ribosome entry sites are found in multiple genera of the family Picornaviridae
More LessThe internal ribosome entry site (IRES) elements from porcine enterovirus 8 and simian virus 2, two members of a proposed new genus within the family Picornaviridae, were characterized. These IRES elements, in common with the porcine teschovirus 1 IRES, were found to be related functionally and structurally to the IRES element from Hepatitis C virus, a member of the family Flaviviridae. Partial secondary structure predictions were derived and functional assays demonstrated that these IRES elements continued to be active when eIF4G was cleaved and when the activity of eIF4A was blocked.
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Deletions in the hypervariable domain of the nsP3 gene attenuate Semliki Forest virus virulence
More LessMajor virulence determinants of Semliki Forest virus (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5′ conserved domains and a non-essential hypervariable 3′ domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.
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Molecular tracing of Japan-indigenous hepatitis E viruses
The ancestor(s) of apparently Japan-indigenous strains of Hepatitis E virus (HEV) was probably of foreign origin, but it remains unclear when and from where it made inroads. In this study, 24 genotype 3 and 24 genotype 4 HEV strains recovered in Japan each showed a significant cluster, clearly distinct from those of foreign strains, in the phylogenetic tree constructed from an 821 nt RNA polymerase gene fragment. The evolutionary rate, approximately 0·8×10−3 nucleotide substitutions per site per year, enabled tracing of the demographic history of HEV and suggested that the ancestors of Japan-indigenous HEV had made inroads around 1900, when several kinds of Yorkshire pig were imported from the UK to Japan. Interestingly, the evolutionary growth of genotype 3 in Japan has been slow since the 1920s, whereas genotype 4 has spread rapidly since the 1980s. In conclusion, these data suggest that the indigenization and spread of HEV in Japan were associated with the popularization of eating pork.
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H5N1 influenza virus evolution: a comparison of different epidemics in birds and humans (1997–2004)
The selection pressure acting along the entire genome sequence of H5N1 avian influenza viruses isolated from several bird species and humans infected in the 1997 and 2004 outbreaks, and on the HA1 genes from H5N1 viruses isolated during the entire study period, in eastern Asia was evaluated. According to maximum-likelihood analysis, viral genes appeared to be, in both epidemics, under strong purifying selection, with only the PB2, HA and NS1 genes under positive selection. Specific codons under positive selection were detected by using codon-based substitution models. Positive-selection analysis performed on single-codon sites might be helpful in clarifying the driving force of avian and human influenza virus evolution and in selecting specific targets for vaccines and antiviral drugs.
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Replication-dependent fitness recovery of Human immunodeficiency virus 1 harbouring mutations of Asn17 of the nucleocapsid protein
More LessThe genetic stability of attenuated Human immunodeficiency virus 1 (HIV-1) variants harbouring mutations (Gly or Lys) of Asn17, the protease-cleavage site of the proximal zinc finger of the nucleocapsid protein, was studied. All possible codons for the Gly mutants were tested as starting sequences. Long-term replication assays revealed that the mutants were unstable; mutations of Gly17 to Arg, Ala, Ser and Cys, as well as a Lys17Asn reversion, were observed. Replication kinetic assays in H9 cells revealed that the replication of Ala, Ser and Arg mutants was improved substantially compared with the Gly variant; the infectivity of Ala17 and Ser17 viruses was equal to, and that of Arg17 was almost equal to, the infectivity of the wild-type virus. Kinetic analysis of the cleavage of oligopeptides representing the corresponding nucleocapsid-cleavage sites revealed that all mutations improved cleavability, in good agreement with the previously proposed role of nucleocapsid cleavage in HIV-1 replication.
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Quantification of Feline immunodeficiency virus (FIVpco) in peripheral blood mononuclear cells, lymph nodes and plasma of naturally infected cougars
More LessInfection of domestic cats with Feline immunodeficiency virus (FIV) results in a fatal immunodeficiency disease, similar to Human immunodeficiency virus 1 (HIV-1) in humans. Elevated plasma viral loads in domestic cats are correlated to decreased survival time and disease progression. However, FIV is also maintained as an apathogenic infection in other members of the family Felidae including cougars, Puma concolor (FIVpco). It is not known whether the lack of disease in cougars is a result of diminished virus replication. A real-time PCR assay was developed to quantify both FIVpco proviral and plasma viral loads in naturally infected cougars. Proviral loads quantified from peripheral blood mononuclear cells (PBMC) ranged from 2·90×101 to 6·72×104 copies per 106 cells. Plasma viral loads ranged from 2·30×103 to 2·81×106 RNA copies ml−1. These data indicate that FIVpco viral loads are comparable to viral loads observed in endemic and epidemic lentivirus infections. Thus, the lack of disease in cougars is not due to low levels of virus replication. Moreover, significant differences observed among cougar PBMC proviral loads correlated to viral lineage and cougar age (P=0·014), which suggests that separate life strategies exist within FIVpco lineages. This is the first study to demonstrate that an interaction of lentivirus lineage and host age significantly effect proviral loads.
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Phylogeny, recombination and expression of porcine endogenous retrovirus γ2 nucleotide sequences
More LessEndogenous retroviral sequences in the pig genome represent a potential infectious risk in xenotransplantation. Porcine endogenous retrovirus (PERV) γ sequences described to date have been classified into several families. The known infectious, human-tropic PERVs have been assigned to the PERV γ1 subfamilies A, B and C. High copy numbers and full-length clones have also been observed for an additional family, designated PERV γ2. The aim of this study was to examine the PERV γ2 family by analysis of retroviral pro/pol gene sequences. The proviral load was observed to be similar among various pig breeds. Although clones harbouring an open reading frame in the examined region were found, analysis of published large PERV γ2 clones revealed multiple deleterious mutations in each of the retroviral genes. Various recombination events between γ2 genomes were revealed. In contrast to PERV γ1, phylogenetic analyses did not distinguish defined subfamilies, but indicated the independent evolution of the proviruses after a single event of retroviral amplification. Expression analysis showed large PERV γ2 transcripts and variable transcription in several tissues. Analysis of the two published γ2 env gene sequences observed the partial lack of the receptor-binding domain. Overall, this study indicated the low infectious potential for PERV γ2.
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Purification and characterization of infectious myonecrosis virus of penaeid shrimp
More LessThe causative agent of myonecrosis affecting cultured Penaeus vannamei in Brazil was demonstrated to be a virus after purification of the agent from infected shrimp tissues. Purified viral particles were injected into specific pathogen-free P. vannamei, resulting in a disease that displayed the same characteristics as those found in the original shrimp used for purification. The virus was named infectious myonecrosis virus (IMNV). The viral particles were icosahedral in shape and 40 nm in diameter, with a buoyant density of 1·366 g ml−1 in caesium chloride. The genome consisted of a single, double-stranded (dsRNA) molecule of 7560 bp. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 5′ ORF (ORF 1, nt 136–4953) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsRNA-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kDa. The 3′ ORF (ORF 2, nt 5241–7451) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsRNA virus family that infects invertebrate hosts.
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- DNA viruses
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Varicella-zoster virus influences the activities of components and targets of the ERK signalling pathway
More LessVaricella-zoster virus (VZV) is ultimately dependent upon its host cell for replication. To ensure its reproduction, VZV reorganizes various cellular functions by taking advantage of pre-existing signalling pathways. Recently, it was demonstrated that the activation of stress-related mitogen-activated protein kinase pathways following infection led to increased phosphorylation of cellular transcription factors involved in VZV gene expression. Here, it was shown that members of the extracellular signal-regulated kinase (ERK) pathway are also influenced following VZV infection: c-Raf remained inactive in infected MeWo cells, whereas MEK1/2 and ERK1/2 were phosphorylated transiently, reaching their highest level of phosphorylation at between 10 and 12 h post-infection. Inhibition of this pathway resulted in a severe reduction in viral progeny and in an increased apoptotic response, indicating that the functionality of this cascade is essential for successful high-rate replication. In addition, the activities of Bad, a cytoplasmic target of ERK via ribosomal S6 kinase, and the nuclear-localized target c-Myc were analysed. Bad is a member of the Bcl-2 family and has a key function in regulating apoptosis. Pro-apoptotic functions of Bad are repressed by phosphorylation. A 10-fold increase in Bad phosphorylation at Ser-112 was detected following infection, which was suppressed after inhibition of ERK. The transcription factor c-Myc is involved in the regulation of cell growth and apoptosis. By performing immunoblots and quantitative RT-PCR, suppression of c-Myc expression was demonstrated at both the transcriptional and translational levels in VZV-infected cells. These results suggest that VZV optimizes the conditions for its replication in different ways: upregulation of proviral-acting systems and suppression of potentially antiviral-acting systems.
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Herpes simplex virus type 1 glycoprotein L mutants that fail to promote trafficking of glycoprotein H and fail to function in fusion can induce binding of glycoprotein L-dependent anti-glycoprotein H antibodies
More LessThe herpes simplex virus type 1 (HSV-1) glycoproteins H (gH) and L (gL) form a heterodimer and efficient expression of gH at the virion or cell surface is dependent upon gL. Five carboxy-terminal deletion mutants of gL were created and their ability to interact with and mediate cell-surface expression of gH, to promote binding of gL-dependent anti-gH antibodies and to contribute to cell fusion was analysed. All of the gL mutants bound gH, but only two mutants, containing the amino-terminal 161 or 168 aa of gL, mediated cell-surface expression of gH, and only gL161 and gL168 functioned in cell fusion. The binding of gL to gH, therefore, was not sufficient to ensure gH cell-surface expression and it was not possible to separate the gH-trafficking role of gL from gL function in fusion. Co-expression of gH with any gL mutant conferred binding of the anti-gH mAbs 53S and LP11. If the acquisition of 53S and LP11 binding to gH reflects a gL-induced conformational change, such a change is not sufficient to mediate trafficking of the gH–gL heterodimer.
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Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek's disease
Herpesvirus of turkey (HVT) is an alphaherpesvirus that is widely used as a live vaccine against Marek's disease because of its antigenic relationship with Marek's disease virus (MDV). In spite of a similar genome structure, HVT has several unique genes, the functions of which are not completely understood. As a first step in carrying out detailed analysis of the functions of the HVT genes, a full-length infectious bacterial artificial chromosome (BAC) clone of HVT was constructed. DNA from two independent BAC clones, upon transfection into chicken embryo fibroblasts, produced plaques similar to those produced by the wild-type virus. Viruses derived from the BAC clones were stable during in vitro passage, but showed differences in in vitro growth kinetics compared with the wild-type virus. Using a one-step mutagenesis protocol to delete the essential glycoprotein B gene from the HVT genome, followed by construction of the revertant virus, BAC clones of HVT were shown to be amenable to standard mutagenesis techniques. In spite of the difference in in vitro growth, viruses from both clones induced 100 % protection against infection by the virulent MDV strain RB-1B, indicating that the BAC-derived viruses could be used as vaccines with efficacies similar to that of the parental virus. The construction of HVT BAC is a major step in understanding the functions of HVT genes by exploiting the power of BAC technology. Furthermore, the availability of the BAC clones enables use of HVT as a vector for expressing foreign genes.
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Characterization and immunological analysis of the rhesus cytomegalovirus homologue (Rh112) of the human cytomegalovirus UL83 lower matrix phosphoprotein (pp65)
More LessRhesus cytomegalovirus (RhCMV) contains two open reading frames (Rh111 and Rh112) that encode proteins homologous to the phosphoprotein 65 (pp65) of the human cytomegalovirus (HCMV) UL83 gene. As HCMV pp65 elicits protective immune responses in infected humans and represents an important vaccination target, one RhCMV homologue of HCMV pp65, pp65-2 (Rh112), was characterized and analysed for its ability to induce host immune responses. Similar to its HCMV counterpart, RhCMV pp65-2 was expressed as a late gene, localized to the nucleus within pp65-2-expressing cells and was present within infectious virions. Longitudinal and cross-sectional studies of pp65-2 immunity in naturally infected rhesus macaques showed that humoral responses to pp65-2 were elicited early during infection, but were not always sustained over time. In contrast, pp65-2-specific T-cell responses, examined by gamma interferon ELISPOT, were broadly detectable in all of the animals studied during primary infection and persisted in the vast majority of RhCMV-seropositive monkeys. Moreover, there was considerable inter-animal variability in the pattern of the immune responses to pp65-2. Together, these results demonstrated that RhCMV pp65-2 exhibited biological and immunological homology to HCMV pp65. Thus, the rhesus macaque model of HCMV persistence and pathogenesis should be relevant for addressing pp65-based vaccine modalities.
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Nuclear localization of the Epstein–Barr virus EBNA3B protein
More LessThe Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.
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