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Volume 82,
Issue 9,
2001
Volume 82, Issue 9, 2001
- Animal: DNA Viruses
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Characterization of a novel genital human papillomavirus by overlapping PCR: candHPV86 identified in cervicovaginal cells of a woman with cervical neoplasia
More LessA novel human papillomavirus (HPV), candHPV86, was cloned and characterized from cervicovaginal cells obtained from a 37-year-old Hispanic woman with cervical intraepithelial neoplasia grade 1 (CIN1) using an overlapping PCR technique. Primers were designed by phylogenetic alignment of closely related HPV genomes using the L1 fragment sequence amplified by GP5+/6+. The 7983 bp complete nucleotide sequence of the HPV genome was determined by sequence walking. A basic local alignment sequence tool (BLAST) homology search using the L1 open reading frame demonstrated that this HPV was most closely related to HPVHAN2294 (GenBank, AJ400628; 86% homology) and HPV84 (84% homology). candHPV86 was placed in the HPV genome homology group A3 by phylogenetic analyses. The overlapping PCR technique is applicable for characterizing the complete spectrum and variation of HPVs in a population.
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Genomic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTVs in humans and non-human primates
TT virus (TTV) was recovered from the sera of tupaias (Tupaia belangeri chinensis) by PCR using primers derived from the noncoding region of the human TTV genome, and its entire genomic sequence was determined. One tupaia TTV isolate (Tbc-TTV14) consisted of only 2199 nucleotides (nt) and had three open reading frames (ORFs), spanning 1506 nt (ORF1), 177 nt (ORF2) and 642 nt (ORF3), which were in the same orientation as the ORFs of the human prototype TTV (TA278). ORF3 was presumed to arise from a splicing of TTV mRNA, similar to reported human TTVs whose spliced mRNAs have been identified, and encoded a joint protein of 214 amino acids with a Ser-, Lys- and Arg-rich sequence at the C terminus. Tbc-TTV14 was less than 50% similar to previously reported TTVs of 3·4–3·9 kb and TTV-like mini viruses (TLMVs) of 2·8–3·0 kb isolated from humans and non-human primates, and known animal circoviruses. Although Tbc-TTV14 has a genomic length similar to animal circoviruses (1·8–2·3 kb), Tbc-TTV14 resembled TTVs and TLMVs with regard to putative genomic organization and transcription profile. Conserved motifs were commonly observed in the coding and noncoding regions of the Tbc-TTV14 genome and in all TTV and TLMV genomes. Phylogenetic analysis revealed that Tbc-TTV14 is the closest to TLMVs, and is closer to TTVs isolated from tamarin and douroucouli than to TTVs isolated from humans and chimpanzees. These results indicate that tupaias are naturally infected with a new TTV species that has not been identified among primates.
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A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors
The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses – a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene – produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.
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Efficient downregulation of major histocompatibility complex class I molecules in human epithelial cells infected with cytomegalovirus
More LessLiver and intestinal epithelial cells are a major target of infection by cytomegaloviruses (CMV), causing severe disease in affected organs of immunocompromised patients. CMV downregulates major histocompatibility complex class I (MHC-I) molecule expression in fibroblasts in order to avoid lysis by CD8+ cytotoxic T lymphocytes. However, MHC-I expression in human cytomegalovirus (HCMV)-infected hepatic tissue was reported to be increased. As it is unclear at present whether HCMV affects MHC-I expression in epithelial cells, new cell culture models for HCMV infection of differentiated hepatobiliary cell lines were established. HCMV immediate early gene expression was achieved in 60 to 95% of cells. Progression of the HCMV replication cycle differed from prototypic infection of fibroblasts, since structural early and late proteins were produced at low levels and HCMV progeny yielded much lower titres in hepatobiliary cells. In contrast, HCMV glycoproteins, gpUS2, gpUS3, gpUS6 and gpUS11, that downregulate MHC-I expression were synthesized with temporal kinetics and in a similar quantity to that seen in fibroblasts. As a result, HCMV infection led to a drastic and selective downregulation of MHC-I expression in epithelial cells and was uniformly observed irrespective of the hepatic or biliary origin of the cells. The new models document for the first time a stealth function of HCMV in epithelial cells and indicate that the downregulation of MHC-I expression by HCMV can occur in the virtual absence of virus replication.
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The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1
The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1vhs− deletion mutant. These results indicate that the BHV1 UL41 gene product is both necessary and sufficient for shutoff of host cell protein synthesis at early times post-infection. Using the vhs deletion mutant, we investigated the mechanism of BHV1-induced down-regulation of MHC class I cell surface expression. In contrast to BHV1 wild-type infection, the BHV1vhs− mutant allows detection of MHC class I molecules at much later time-points after infection. This illustrates the role the vhs protein plays in MHC class I down-regulation. However, even after infection with BHV1vhs−, MHC class I cell surface expression is impaired. In BHV1vhs−-infected cells, MHC class I molecules are retained within the endoplasmic reticulum (ER). Moreover, the transporter associated with antigen presentation (TAP) is still blocked. Temporal control of viral protein expression using chemical inhibitors shows that viral protein(s) expressed within the early phase of BHV1 infection are responsible for ER retention of MHC class I molecules. These results indicate that multiple mechanisms are responsible for down-regulation of MHC class I molecules in BHV1-infected cells.
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Vaccinia virus semaphorin A39R is a 50–55 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model
More LessVaccinia virus (VV) protein A39R has amino acid similarity to the extracellular domain of a glycosylphosphatidylinositol-linked cell surface semaphorin (SEMA7A/CDw108) that has an immunological expression profile and binding properties, thereby implicating A39R as an immunomodulator. Previously, a closely related A39R protein expressed by ectromelia virus was shown to induce cytokine production and up-regulate ICAM-1 expression in mouse monocytes in vitro. In this study, we show that the A39R gene of VV strain Copenhagen (COP) encodes a 50–55 kDa secreted glycoprotein and is expressed late during infection. The A39R protein was secreted by eight of 15 strains of VV, but not by strain Western Reserve (WR). To analyse the VV A39R function, several recombinant viruses were made, including an A39R deletion mutant of VV COP and a WR mutant containing the A39R sequence from COP. Loss of the gene from COP did not affect virus growth in vitro, or VV virulence in a mouse intranasal model, and had only a slight effect on lesion size in an intradermal model. In contrast, expression of COP A39R by VV WR was associated with an increase in the severity and persistence of skin lesions after intradermal infection of mice. Finally, a histological examination of mouse skin infected with recombinant viruses suggested that A39R has direct or indirect pro-inflammatory properties.
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The vaccinia virus A41L protein is a soluble 30 kDa glycoprotein that affects virus virulence
More LessVaccinia virus (VV) gene A41L encodes an acidic protein with amino acid similarity to the 35 kDa protein of VV strain Lister, a soluble protein called vCKBP that binds CC chemokines, and to a protein from orf virus, called GIF, that binds GM-CSF and IL-2. However, despite the similarity, recombinant A41L protein was found not to bind these ligands or a variety of other chemoattractant molecules when tested using surface plasmon resonance. The A41L gene is expressed early and late during infection and encodes a 30 kDa protein that contains both N- and O-linked carbohydrate and is secreted from the infected cell. All 16 strains of VV and 2 strains of cowpox virus that were tested express the A41L protein, implying it has an important function for orthopoxviruses. Nonetheless, a VV strain Western Reserve deletion mutant lacking the A41L gene (vΔA41L) formed normal sized plaques and replicated to the same titre as wild-type and revertant viruses. The importance of the A41L protein in vivo was demonstrated in a mouse intradermal model in which infection with vΔA41L caused more severe lesions compared to wild-type and revertant viruses. Further examination in this model revealed that deletion of A41L enhanced clearance of infectious virus, suggesting that A41L expression reduces immunopathology. Consistent with this, histological examination of infected rabbit skin showed that the A41L protein could reduce the infiltration of inflammatory cells into the infected area. Together, these data suggest that the A41L protein constitutes a novel immunomodulatory protein.
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Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine
More LessRecombinant vaccinia viruses are well-characterized tools that can be used to define novel approaches to vaccine formulation and delivery. While vector co-expression of immune mediators has enormous potential for optimizing the composition of vaccine-induced immune responses, the impact on antigen expression and vector antigenicity must also be considered. Co-expression of IL-4 increased vaccinia virus vector titres, while IFN-γ co-expression reduced vaccinia virus replication in BALB/c mice and in C57BL/6 mice infected with some recombinant viruses. Protection against respiratory syncytial virus (RSV) challenge was similar in mice immunized with vaccinia virus expressing RSV G glycoprotein and IFN-γ, even though the replication efficiency of the vector was diminished. These data demonstrate the ability of vector-expressed cytokine to influence the virulence of the vector and to direct the development of selected immune responses. This suggests that the co-expression of cytokines and other immunomodulators has the potential to improve the safety of vaccine vectors while improving the immunogenicity of vaccine antigens.
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- Insect
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A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag
More LessThe Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74–GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74–GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74–GFP chimeras in recombinant baculoviruses. When C-terminal region S580–F645 was deleted from p74, p74–GFP chimera localization became non-specific and chimeras became soluble. p74 region S580–F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.
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Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells
More LessThe Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1·5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5′ upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3′ end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.
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Phylogenetic analysis of conserved genes within the ecdysteroid UDP-glucosyltransferase gene region of the slow-killing Adoxophyes orana granulovirus
More LessA physical map of the genome of Adoxophyes orana granulovirus (AoGV) was constructed for the restriction enzymes BamHI, BglII, EcoRI, PstI and SacI using restriction endonuclease analysis and DNA hybridization techniques. This enabled the size of the AoGV genome to be estimated at 100·9 kbp. A plasmid library covering 99·9% of the AoGV genome was constructed using five restriction enzymes. The ecdysteroid UDP-glucosyltransferase gene (egt) was located by hybridization with the egt gene of Cydia pomonella granulovirus. The sequence of 6000 bp of the egt region is presented and compared to the equivalent area in other GVs. Database searches showed that this region contained eight open reading frames (ORFs) similar to the baculovirus genes egt, granulin, pk-1, me53 and four ORFs of Xestia c-nigrum granulovirus (ORF 178, ORF 2, ORF 7 and ORF 8). The egt gene was shown to encode an active EGT using an EGT assay. Phylogenetic trees of the granulovirus genes egt, granulin, pk-1 and me53 were constructed using maximum parsimony and distance analyses. These analyses indicated that AoGV genes may be more closely related to other tortricid-infecting GVs than to GVs that infect other lepidopteran families.
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- Plant
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Coat protein gene-mediated resistance to Potato virus A in transgenic plants is suppressed following infection with another potyvirus
More LessHigh levels of resistance to Potato virus A (PVA, genus Potyvirus), indicated by absence of detectable infection in inoculated leaves, were attained in Nicotiana benthamiana transformed with a construct expressing the PVA 5′-untranslated region fused with the coat protein (CP)-encoding sequence. Low steady-state levels of the transgene transcripts were detected. Resistance was PVA-specific and did not protect the plants against infection with Potato virus Y (PVY, genus Potyvirus). Consequently, the steady-state levels of the CP-transgene mRNA were greatly elevated in the plants infected with PVY, and plants became susceptible to infection with PVA. These data show that virus resistance obtained by expressing regions of a plant virus genome in transgenic plants may be suppressed following infection with another virus that evades the virus-specific resistance.
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