Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus

Rapp, Maria; Messerle, Martin; Bühler, Bernhard; Tannheimer, Michael; Keil, Günther M.; Koszinowski, Ulrich H.

doi:10.1128/jvi.66.7.4399-4406.1992

Cited by 78 publications

(29 citation statements)

References 38 publications

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“…Because this sequence does not encompass a splice donor motif (23), we concluded that insufficient stability of the heteroduplexes due to AT-rich sequences around the poly(A) signal (Fig. 2) resulted in overdigestion of the DNA-RNA hybrids (25). Thus, the exact 3Ј end of the BHV-1 UL6 and UL7 mRNAs remains to be determined, as does the corresponding poly(A) addition site for HSV-1 (24).…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture

Schmitt

Keil

1996

J Virol

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The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3 codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture.

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Section: Discussionmentioning

confidence: 99%

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture

Schmitt

Keil

1996

J Virol

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“…As a source of viral antigens, lysates of infected MEF were prepared 24 h after infection after a 4-h period of metabolic labeling with [3SS]methionine in the late phase of viral gene expression. Lysates from CV-1 cells were made 7 h after infection with vaccinia virus wild-type strain Copenhagen or with the vaccinia virus recombinant Vac-gB which expresses the gB gene of murine CMV (15). Procedures for biosyn-thetical labeling, preparation of cell lysates, and the immunoprecipitation as well as the protein separation on SDS-polyacrylamide (7.5%) gels were done as described previously (15,16).…”

Section: Methodsmentioning

confidence: 99%

The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease.

Reddehase

Balthesen

Rapp

et al. 1994

The Journal of Experimental Medicine

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196

199

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SummaryRecurrence of cytomegalovirus (CMV) from latency is a frequent cause of disease in immunocompromised patients. To date, there is no explanation for the diversity in the clinical manifestations. Primary infection can occur perinatally or later in life, and inevitably results in latent infection. Seropositivity for antibodies against CMV is indicative of latent infection, but is insufficient as a predictor for the risk of recurrence. As a model for this important medical problem, we compared the risks of murine CMV recurrence from latency established after neonatal primary infection and after infection at adult age. The risk of CMV recurrence was high only after neonatal infection. The copy number of latent viral genome in tissues was identified as the key parameter that determines the overall and organ-specific risks of recurrence. Latent CMV burden and risk of recurrence were related to the extent of virus multiplication during primary infection. The presence of latent CMV in multiple organs provides the molecular basis for stochastic events of recurrence in single organs or in any combination thereof. These findings are discussed as a concept of multifocal CMV latency and recurrence. It provides a rationale for the diversity in the clinical outcome of CMV disease.

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“…Strict species specificity has hindered the study of human CMV (HCMV) in animals, and infection of mice with murine CMV (MCMV) has been used extensively as a model for studying the pathogenesis of CMV infection (8,34,46,57). The human and murine viruses are biologically similar in replication and pathogenesis (25,45) and have homologous genomes (52), which display similar genetic organizations and encode analogous gene products with similar functions (19,31,43,44,50,51,68). The genomes of HCMV and MCMV are linear, double-stranded DNA molecules approximately 230 kb in length and encode more than 200 genes (10,52), many of which are arranged collinearly on the HCMV and MCMV genomes.…”

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Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism

Cavanaugh

Stenberg

Staley

et al. 1996

J Virol

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Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.Cells. Murine NIH 3T3 fibroblasts (American Type Culture Collection [ATCC] CRL1658; ATCC, Rockville, Md.) were propagated in Dulbecco's modified essential medium (DMEM; Mediatech, Herndon, Va.) supplemented with 10% heat-inactivated bovine calf serum (HyClone Laboratories, Logan, Utah) and 1% L-glutamine (Gibco/BRL, Grand Island, N.Y.). IC-21 cells, a simian virus 40-transformed peritoneal macrophage cell line derived from C57BL/6 mice (41), were obtained from ATCC and were propagated in RPMI medium (Mediatech) supplemented with 10% heat-inactivated fetal calf serum (Gibco/BRL) and 1% L-glutamine. Primary fibroblasts, derived from the embryos of timedpregnant BALB/c.ByJ mice (Jackson Laboratory, Bar Harbor, Maine) on day 19 of gestation, were cultured in DMEM containing 20% heat-inactivated fetal calf serum, 1% L-glutamine, and 50 g of gentamicin sulfate (Sigma Chemical Co., St. Louis, Mo.) per ml.Virus. The parental WT MCMV used in this study was of the Smith strain (ATCC VR-194). Stocks of WT and mutant viruses were prepared in and titers were determined on NIH 3T3 cells as previously described (8). Mock preparations of virus consisted of culture supernatants from noninfected NIH 3T3 cells.Mice. BALB/c.ByJ mice, either 20-day-old weanlings (...

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Identification of the murine cytomegalovirus glycoprotein B gene and its expression by recombinant vaccinia virus

Cited by 78 publications

References 38 publications

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture

The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease.

Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism

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