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At least 1,279 records · Page 71Linked to original sources
PubMed1988

Mapping of the fibrotropic and lymphotropic host range determinants of the parvovirus minute virus of mice.

The fibrotropic and lymphotropic strains of minute virus of mice are each unable to grow lytically in the differentiated host cell type of the other strain. To map the viral sequence responsible for the target cell specificities of the two strains, we constructed chimeric viral genomes in vitro from infectious genomic clones. The phenotypes of viral progeny derived from the chimeric genomes were tested by transfecting the plasmids into fibroblast monolayers and assaying plaque formation and by testing stocks of the recombinant viruses for cytotoxicity in fibroblast and lymphocyte cultures. Both the fibrotropic and lymphotropic determinants mapped to the same 237-nucleotide sequence within the coding region of the virus structural gene. A second sequence, near the viral promoter at map unit 38, was also shown to affect viral growth in fibroblast host cells profoundly.

Animals↗
PubMed1990

Synthesis of hepadnavirus particles that contain replication-defective duck hepatitis B virus genomes in cultured HuH7 cells.

To evaluate the possibility of producing transducible replication-defective hepadnaviruses, cloned mutant duck hepatitis B virus genomes were tested both for virus antigen production and viral DNA synthesis following transfection into the human hepatoma cell line HuH7. Deletion of a cis-acting 12-nucleotide sequence implicated in viral DNA synthesis, direct repeat 1 (DR1), resulted in the loss of ability to synthesize both mature viral DNA and infectious virus. The delta DR1 mutant, however, produced envelope and core antigens and was shown to provide trans-acting functions required for the assembly of infection-competent particles. Thus, mutants with mutations in viral genes could be rescued as DNA-containing viral particles after cotransfection with delta DR1. The efficiency of rescue was influenced by the site of mutation. A mutant DNA encoding truncated core and envelope proteins not only was poorly rescued but also was able to suppress the production from a wild-type DNA of infectious virus.

Animals↗
PubMed1992

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis.

Cell-Free System↗
PubMed1992

Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1.

Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different herpesvirus. Our data show that in gII- PrV, gI(BHV-1) in vitro fully complements the lethal defect associated with lack of gII(PrV). These results conclusively demonstrate that gI(BHV-1) in a PrV background can execute all essential functions normally provided by gII(PrV). They also indicate that the origin of gB-homologous glycoproteins influences the penetration kinetics of herpesviruses.

Animals↗
PubMed1994

Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates.

Mouse hepatitis virus (MHV) A59 temperature-sensitive (ts) mutants belonging to complementation group C were characterized and mapped by standard genetic recombination techniques. Temperature shift experiments early in infection suggested that the group C allele can be divided into two phenotypically distinct subgroups, designated C1 and C2. Since previous data indicated that the group C1 mutants probably contained an early defect which affects negative-strand synthesis, RNA synthesis was further examined by analyzing replicative-form (RF) RNA. Full-length as well as subgenomic-length RF RNAs were radiolabeled from 3 to 12 h postinfection (p.i.) and labeled late in infection after shift to the nonpermissive temperature (39.5 degrees C). The relative percent molar ratios of each mRNA and corresponding RF RNA were roughly equivalent throughout infection. Temperature shift experiments at 5.5 or 6.0 h p.i. resulted in an 83 to 92% reduction in the amount of total RF RNA at 39.5 degrees C. Radiolabeling time course experiments after temperature shift to 39.5 degrees C also demonstrated incorporation (6 to 9 h p.i.) into both subgenomic-length and full-length RF RNAs, suggesting that previously transcribed negative strands were functional templates throughout infection. To determine if the reduction in RF RNA was due to a decrease in positive- or negative-strand RNA synthesis, rates of mRNA synthesis were calculated from both full-length and subgenomic-length templates. The rate of mRNA synthesis after the shift was increased at 39.5 degrees C compared with that at 32 degrees C regardless of the template used; however, transcription rates calculated from subgenomic-length templates were similar to those of other viral and eukaryotic polymerases. These findings support the notion that the group C1 allele regulates negative-strand RNA synthesis and strongly suggest that the subgenomic negative-strand RNAs are probably the predominant functional templates for the synthesis of positive-strand RNAs late in infection.

Animals↗
PubMed1998

Hepatitis B virus X protein interferes with cellular DNA repair.

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell's capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.

Animals↗
PubMed1998

Enhancer requirement for murine cytomegalovirus growth and genetic complementation by the human cytomegalovirus enhancer.

The cytomegalovirus (CMV) enhancer is a highly complex regulatory region containing multiple elements that interact with a variety of host-encoded transcription factors. Many of these sequence elements are conserved among the different species strains of CMV, although the arrangement of the various elements and overall sequence composition of the CMV enhancers differ remarkably. To delineate the importance of this region to a productive infection and to explore the possibility of generating a murine CMV (MCMV) under the control of human CMV (HCMV) genetic elements, the MCMV enhancer was resected and replaced either with nonregulatory sequences or with paralogous sequences from HCMV. The effects of these various deletions and substitutions on viral growth in transfected or infected tissue-culture cells were evaluated. We found that mutations in MCMV that eliminate or substitute for the enhancer with nonregulatory sequences showed a severe deficiency in virus synthesis. This growth defect is effectively complemented by the homologous MCMV enhancer as well as the HCMV enhancer. In the latter case, the chimeric viruses (hybrid MCMV strains) containing the molecularly shuffled human enhancer exhibit infectious kinetics similar to that of parental wild-type and wild-type revertant MCMV. These results also show that open reading frames m124, m124.1, and m125 located within the enhancer region are nonessential for growth of MCMV in cells. Most importantly, we conclude that the enhancer of MCMV is required for optimal infection and that its diverged human counterpart can advantageously replace its role in promoting viral infectivity.

3T3 Cells↗
PubMed2000

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system.

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.

Animals↗
PubMed2004

The DNA-A component of a plant geminivirus (Indian mung bean yellow mosaic virus) replicates in budding yeast cells.

Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.

Base Sequence↗
PubMed2006

Functional domains of murine cytomegalovirus nuclear egress protein M53/p38.

Two conserved herpes simplex virus 1 proteins, UL31 and UL34, form a complex at the inner nuclear membrane which governs primary envelopment and nuclear egress of the herpesvirus nucleocapsids. In mouse cytomegalovirus, a member of the betaherpesvirus subfamily, the homologous proteins M53/p38 and M50/p35 form the nuclear egress complex (NEC). Since the interaction of these proteins is essential for functionality, the definition of the mutual binding sites is a prerequisite for further analysis. Using a comprehensive random mutagenesis procedure, we have mapped the M53/p38 binding site of M50/p35 (A. Bubeck, M. Wagner, Z. Ruzsics, M. Lötzerich, M. Iglesias, I. R. Singh, and U. H. Koszinowski, J. Virol. 78:8026-8035). Here we describe a corresponding analysis for the UL31 homolog M53/p38. A total of 72 individual mutants were reinserted into the genome to test the complementation of the lethal M53 null phenotype. The mutants were also studied for colocalization and for coprecipitation with M50/p35. The analysis revealed that the nonconserved N-terminal one-third of M53/p38 provides the nuclear localization signal as an essential function. The collective results for many mutants localized the binding site for M50/p35 to amino acids (aa) 112 to 137. No single aa exchange for alanine could destroy NEC formation, but virus attenuation revealed a major role for aa K128, Y129, and L130. The lethal phenotype of several insertion and stop mutants indicated the functional importance of the C terminus of the protein.

Animals↗
PubMed1993

Two novel transmembrane protein tyrosine kinases expressed during Caenorhabditis elegans hypodermal development.

We describe our characterization of kin-15 and kin-16, a tandem pair of homologous Caenorhabditis elegans genes encoding transmembrane protein tyrosine kinases (PTKs) with an unusual structure: the predicted extracellular domain of each putative gene product is only about 50 amino acids, and there are no potential autophosphorylation sites in the C-terminal domain. Using lacZ fusions, we found that kin-15 and kin-16 both appear to be expressed during postembryonic development in the large hypodermal syncytium (hyp7) around the time that specific hypodermal cells fuse with hyp7. kin-15 and kin-16 were positioned on the genetic and physical maps, but extrachromosomal arrays containing wild-type kin-15 and/or kin-16 genes were unable to complement candidate lethal mutations. The results suggest that kin-15 and kin-16 may be specifically involved in cell-cell interactions regulating cell fusions that generate the hypodermis during postembryonic development.

Amino Acid Sequence↗
PubMed1995

Six human RNA polymerase subunits functionally substitute for their yeast counterparts.

To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.

Amino Acid Sequence↗
PubMed2005

Fen-1 facilitates homologous recombination by removing divergent sequences at DNA break ends.

Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.

Animals↗
PubMed2005

Cost effectiveness of a new strategy to identify HNPCC patients.

BACKGROUND: Distinguishing hereditary non-polyposis colorectal cancer (HNPCC) from non-hereditary colorectal cancer (CRC) can increase the life expectancy of HNPCC patients and their close relatives. AIM: To determine the effectiveness, efficiency, and feasibility of a new strategy for the detection of HNPCC, using simple criteria for microsatellite instability (MSI) analysis of newly detected tumours that can be applied by pathologists. Criteria for MSI analysis are: (1) CRC before age 50 years; (2) second CRC; (3) CRC and HNPCC associated cancer; or (4) adenoma before age 40 years. METHODS: The efficacy and cost effectiveness of the new strategy was evaluated against current practice. Decision analytic models were constructed to estimate the number of extra HNPCC mutation carriers and the costs of this strategy. The incremental costs and gain in life expectancy for a HNPCC mutation carrier were evaluated by Markov modelling. Feasibility was explored in five hospitals. RESULTS: Using the new strategy, 2.2 times more HNPCC patients can be identified among a CRC population compared with current practice. This new strategy was found to be cost effective with an expected cost effectiveness ratio of 3801 per life year gained. When including the group of siblings and children, the cost effectiveness ratio became 2184 per life year gained. Sensitivity analysis showed these findings to be robust. CONCLUSIONS: MSI testing in a selection of newly diagnosed CRC patients was shown to be cost effective and a feasible method to identify patients at risk for HNPCC who are not recognised by family history.

Colorectal Neoplasms↗