- Volume 76, Issue 12, 1995
Volume 76, Issue 12, 1995
- Animal
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Development and characterization of a novel xenograft model permissive for human papillomavirus DNA amplification and late gene expression
Human papillomaviruses (HPVs) are important human pathogens associated with a range of epithelial neoplasia. The rising incidence of HPV infection and association of HPV with malignancy has led to increased interest in appropriate management of these infections. Development of new therapies for viral warts has been frustrated by the lack of availability of models permissive for viral replication. Here we describe the development of a HPV-severe combined immunodeficient mouse model which reproduces mature HPV-infected epithelia. Grafting of anogenital and laryngeal papillomas harbouring either HPV-6 or HPV-11 resulted in the formation of a differentiated neo-epithelium exhibiting the hallmark features of HPV infection including basal hyperplasia, acanthosis and koilocytosis. The reformed warty epithelium contained amplified HPV DNA and expressed capsid protein in the differentiated layers. A striking feature is the production of macroscopic papillomata in an anatomically relevant and accessible site, providing a system of particular relevance for the temporal evaluation of developing lesions and selection of antiviral agents.
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The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons
More LessNeural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.
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Thermosensitive UL9 gene function is required for early stages of herpes simplex virus type 1 DNA synthesis
More LessDNA replication of herpes simplex virus type 1 (HSV-1) is dependent on a virus-encoded sequence-specific origin-binding protein, the product of the UL9 reading frame. We have identified the mutations in the UL9 gene of three temperature-sensitive (ts) mutants of HSV-1 which are responsible for the ts phenotype (A90T in mutant tsS and V220M in tsR and tsX). The mutations are located in two different conserved helicase sequence motifs of UL9. Two further alterations (I204T and E280D) compared to the published sequence were found in the mutant, revertant and parental wild-type strain 17syn + sequences and therefore seemed to be irrelevant for the ts phenotype. The ts function of the UL9 protein was required at early times during DNA synthesis whereas upward temperature shifts at later times did not considerably inhibit DNA synthesis.
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The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity
More LessThe UL9 protein of herpes simplex virus type 1 binds to specific sequences within the viral origins of DNA replication and also functions as a DNA helicase. The C-terminal 317 amino acids of the 851 residue protein specify sequence-specific binding to the viral origins and the N-terminal 400 contain several motifs characteristic of many DNA and RNA helicases. To investigate whether the origin-binding domain is required for helicase function we have expressed a truncated version comprising amino acids 1-535 of UL9 using a recombinant baculovirus. Extracts were prepared from cells infected with the recombinant virus and chromatographed over ATP-agarose. DNA helicase, DNA-dependent ATPase and a novel single-stranded DNA-binding activity were present in fractions containing the truncated UL9 protein but not in corresponding gradient fractions from a control virus infection. These results indicate that the DNA helicase function of UL9 does not require the presence of the origin-binding domain and suggest that an interaction between the N-terminal domain and distorted or partially single-stranded regions of DNA may play a role in unwinding the origin region.
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The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis
More LessLytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from the cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effects on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated the expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.
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Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice
The objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model. A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector. In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector. Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity. These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of value as a component of an HSV multi-subunit vaccine.
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Co-expression of the Epstein—Barr virus BXLF2 and BKRF2 genes with a recombinant baculovirus produces gp85 on the cell surface with antigenic similarity to the native protein
More LessGlycoprotein H (gH) is a conserved herpesvirus gene product functionally implicated in the penetration of the virus into the host cell. Other human herpesviruses require an accessory glycoprotein named gL for the synthesis of mature gH. We constructed a series of recombinant baculoviruses to determine whether gL expression can overcome the constraints upon Epstein—Barr virus (EBV) gH (gp85) folding and transport in this expression system. When gH and gL were co-expressed some EBV gH was transported to the insect cell surface. Deletion of 27 amino acids from the gH carboxy terminus resulted in the secretion of an 80 kDa protein (gHt) into the culture medium when it was expressed either in the presence or absence of gL and this protein could also be immunoprecipitated with E1D1. In contrast, gL was not secreted into the culture medium. Our results suggest that either co-expression of gH with gL or removal of the predicted transmembrane anchor sequence can overcome some of the constraints upon EBV gH expression in the baculovirus system.
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Replication-defective adenovirus type 5 as an in vitro and in vivo gene transfer vector in chickens
More LessThe capacity of E1A gene-deleted and thus replication-defective adenovirus type 5 (Ad5) to transduce foreign genes in chicken embryo fibroblasts (CEF) as well as in chickens was investigated. The lacZ and luciferase genes were successfully transduced in CEF by replication-defective Ad5, demonstrating that these cells possess receptor(s) for binding and penetration of Ad5. A single intramuscular inoculation of Ad-gD, a replication-defective Ad5 harbouring the gD gene of pseudorabies virus, in adult and 1-day-old chickens led to the production of very high titres of specific antibodies. These gD-specific antibodies persisted for at least 56 days. These results demonstrate that replication-defective Ad5, despite its mammalian origin and the deletion of the E1A gene, is a good candidate for developing non-spreading vaccines in poultry.
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The precore/core promoter mutant (T1762A1764) of hepatitis B virus: clinical significance and an easy method for detection
More LessRecently, a new hepatitis B virus (HBV) mutant with HBe antigen-negative phenotype has been characterized, in which one TATA box-like motif of the precore/core promoter had degenerated: most frequently by both A → T and G → A mutations at positions 1762 and 1764, respectively. The clinical significance of this mutant is as yet unknown. In our present study, the T1762 A1764 mutant was sought in sera from HBV-infected blood donors and chronic liver disease patients by directly sequencing a PCR-amplified region of HBV DNA. Also, because the A1764 mutation generates a Sau3AI cleavage site (GGTC → GATC), we digested the PCR products with Sau3AI to see if cleavage would occur at this specific site. Our results mostly corroborated the earlier report but we found a higher-than-predicted frequency of HBe antigen-positive blood donors positive for the mutant (22%). The titres of HBe antigen in these mutant-positive sera were slightly decreased compared to the titres in wild-type HBV infection. In addition, these blood donors had relatively high (though within the normal range) serum alanine aminotransferase (ALT) levels, suggesting that the T1762 A1764 mutation could be used as a sensitive laboratory marker for insidious hepatitis in these otherwise ‘asymptomatic’ carriers. The Sau3AI assay, which is much more convenient than sequencing, was shown to be useful for the detection of the T1762 A1764 mutant in an extensive number of clinical samples.
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Targeted infection of a retrovirus bearing a CD4-Env chimera into human cells expressing human immunodeficiency virus type 1
More LessWe constructed a hybrid Moloney murine leukaemia virus (MoMLV) bearing both a chimeric CD4 and the wild-type MoMLV envelope protein (Env) on its surface. The chimeric molecule, consisting of a surface domain of CD4 and the C-terminal two-thirds of MLV Env, was expressed on the cell surface. When expressed in MoMLV-infected cells, the CD4-Env chimera was incorporated into the virion as efficiently as the wild-type MoMLV Env. The hybrid MoMLV could infect human HeLa cells (although not with high efficiency) only if the cells were expressing human immunodeficiency virus type 1 genome. This method of ligand incorporation into a virion may lead to a development of a cell-specific retroviral vector for targeting gene therapy.
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Role of the C terminus Gag protein in human immunodeficiency virus type 1 virion assembly and maturation
X.-F. Yu, Z. Matsuda, Q.-C. Yu, T.-H. Lee and M. EssexLentiviral Gag polyproteins have a proline-rich protein, p6, at their C terminus. There are conflicting reports about the function of p6 in virus release. In the present work, mutants that affect p6 of human immuno-deficiency virus type 1 (HIV-1) Gag polyprotein were constructed and analysed. None of the mutants prevented virus release completely; however, detachment of budding particles was less efficient as evidenced by electron microscopy. Virions of the p6 truncation mutant B2TAA had a significantly reduced number of Pol proteins (p66, p51 and p34) and an increased amount of incompletely processed Gag proteins compared with the parental virus. A mutation that altered the cleavage site between p6 and p1 did not significantly affect virus assembly, virus release or protein processing with the exception of cleavage between p6 and p1. However, virions of this mutant (B2P6C) exhibited irregularshaped core structures that were distinct from the cone-shaped core structure seen in the parental virion. B2P6C mutant virus was non-infectious in CD4+ T cells. These results suggest that mutations in p6 affect efficient detachment of budding particles from the cell surface. Proper cleavage between p6 and p1 may be critical for the formation of the distinctive cone-shaped core structure of HIV-1 virions.
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Sequence comparison of open reading frames 2 to 5 of low and high virulence United States isolates of porcine reproductive and respiratory syndrome virus
More LessThe sequences of ORFs 2 to 5 of five United States (US) porcine reproductive and respiratory syndrome virus (PRRSV) isolates with differing virulence were determined. The nucleotide and deduced amino acid sequences of these isolates were compared with those of other known PRRSV isolates. The amino acid sequence identity between seven US PRRSV isolates was 91–99% in ORF 2, 86–98% in ORF 3, 92–99% in ORF 4 and 88–97% in ORF 5. The low virulence US isolate had highest sequence variation in ORFs 2 to 4 compared to the other US isolates. A hypervariable region with antigenic potential was identified within the major envelope glycoprotein. Phylogenetic analysis of ORFs 2 to 7 indicated the existence of at least three minor genotypes within the major US genotype. The low virulence US isolate formed a branch distinct from the other US isolates. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.
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A minimal and optimal cytotoxic T cell epitope within hepatitis C virus nucleoprotein
Amino acid residues 81–100 of the hepatitis C virus (HCV) nucleoprotein contain a cytotoxic T cell epitope that is recognized by cytotoxic T lymphocytes (CTLs) in association with human leukocyte antigen B44. With panels of truncated and overlapping peptides, the minimal and optimal epitope recognized by CTLs was shown to be a 9-mer peptide (residues 88–96). The peptide can stimulate effectively CTLs that are able to recognize endogenously synthesized and processed HCV nucleoprotein.
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Isla Vista virus: a genetically novel hantavirus of the California vole Microtus californicus
Prospect Hill virus (PH) was isolated from a meadow vole (Microtus pennsylvanicus) in 1982, and much of its genome has been sequenced. Hantaviruses of other New World microtine rodents have not been genetically characterized. We show that another Microtus species (the California vole M. californicus) from the United States is host to a genetically distinct PH-like hantavirus, Isla Vista virus (ILV). The nucleocapsid protein of ILV differs from that of PH by 11.1% and a portion of the G2 glycoprotein differs from that of PH by 19.6%. ILV antibodies were identified in five of 33 specimens of M. californicus collected in 1975 and 1994–1995. Enzymatic amplification studies showed that 1975 and 1994–1995 ILV genomes were highly similar. Secondary infection of Peromyscus californicus was identified in Santa Barbara County, California. A long-standing enzootic of a genetically distinct hantavirus lineage is present in California voles.
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Detection of measles virus nucleoprotein mRNA in autopsied brain tissues
More LessBy means of RT–PCR, a portion of measles virus (MV) mRNA encoding nucleoprotein (NP) could be detected in 11 (18%) of 61 brain tissue samples obtained from administrative autopsy cases, who apparently had not suffered from subacute sclerosing panencephalitis (SSPE)-like central nervous system disorders. Most of the brain-derived NP sequences showed significant asynonymous nucleotide substitutions when compared with wild-type MV isolates and SSPE virus. Our present results suggest that MV commonly persists in the human brain without causing apparent clinical symptoms, probably due to decreased virus replication.
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Sequence analyses of human parainfluenza virus type 4A and type 4B fusion proteins
cDNAs encoding human parainfluenza virus type 4A and type 4B (hPIV-4A and -4B) fusion (F) proteins were cloned and sequenced. The predicted amino acid sequences of the F proteins had similar characteristic traits to those reported for the F proteins of other paramyxoviruses. They were more closely related to the F proteins of simian virus 5 (SV5), mumps virus (MuV), hPIV-2 and Newcastle disease virus (NDV) than to the F proteins of hPIV-1, hPIV-3, Sendai virus (SV) and measles virus (MV). In addition, hPIV-4A, hPIV-4B, SV5 and MuV shared a common feature of genomic organization: there was a small ORF between the F and haemagglutinin—neuraminidase (HN)-coding sequences, implying a common ancestry.
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The relative amount of an influenza A virus segment present in the viral particle is not affected by a reduction in replication of that segment
More LessThe principles of influenza A virus replication and packaging are not fully understood. In order to investigate the signals required for these processes we have introduced mutations in the terminal non-coding region of an influenza A virus neuraminidase (NA) gene. Specifically, we have obtained two viruses, NA/X and NA/Y, which produce a reduced amount of NA-specific genomic RNA in infected cells but not in the viral particle. These data indicate that (i) specific signals which affect the amount of RNA in the viral particle are distinct from those required for viral replication and (ii) the amount of packaged RNA is not strictly dependent on the amount of RNA produced during replication. In addition, mutant NA/Y was shown to be effectively attenuated in mice. Thus, diminished replication of one viral segment might be a principle on which to base a live influenza virus vaccine.
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Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed
More LessA full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.
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Differences in the subcellular localization of tobacco mosaic virus and cucumber mosaic virus movement proteins in infected and transgenic plants
More LessOur study reveals differences in the way that tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) movement proteins (MPs) partition with cellular components separated into four fractions from different aged leaves of infected and transgenic plants. Immunoblot analyses showed that TMV and CMV MPs associated predominantly with components in the cell wall fractions from leaves of transgenic plants. In infected tissue, highest levels of TMV MP were found in the organelle fractions from young and middle-aged leaves whereas most of the CMV MP was found in the detergent wash of the cell wall fraction. These results remained consistent even when plants were doubly infected with TMV and CMV. These results imply that MPs of plant viruses from different taxonomic groups differentially associate with subcellular components and that MP produced by a viral infection is targeted to additional subcellular destinations than MP produced in transgenic plants.
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Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus
More LessAn infectious full-length cDNA clone of the RNA genome of the potyvirus zucchini yellow mosaic virus (ZYMV) was constructed under the control of the cauliflower mosaic virus 35S promoter. All squash, cucumber, melon and watermelon plants inoculated with the cloned cDNA of ZYMV by particle bombardment become infected. Bombardment technology is 106-fold more effective than mechanical inoculation. Due to the great increase in efficiency, ineffective constructs now became infective (i.e. cDNA under the control of the 35S promoter without the NOS terminator; with an addition of 127 nucleotides at the 5′ end of the viral cDNA; uncapped transcripts), and the infectivity of capped-transcripts was maximized. Inoculation by particle bombardment produced visual symptoms rapidly (3–4 days), allowing the detection of viral coat protein and virions after 2 and 3 days in systemically infected leaves and inoculated cotyledons respectively.
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Volume 105 (2024)
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