Biomedical subjects

D J Tenney

Publications and source records attributed to D J Tenney.

26 records · Page 2Linked to original sources

PubMed1993

Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain.

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.

Amino Acid Sequence↗

PubMed1992

Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.

Amino Acid Sequence↗

PubMed1991

Expression of the human cytomegalovirus UL36-38 immediate early region during permissive infection.

We have characterized the expression of human cytomegalovirus immediate early (IE) genes encoding the UL36-38 open reading frames. Single-stranded RNA hybridization probes, transcribed from UL36-38 cDNAs or smaller exon-specific clones, were used in analyses of RNA isolated during various phases of infection in the presence and absence of inhibitors of protein and HCMV DNA synthesis. There are three IE transcripts that arise by the use of alternative promoters and splicing signals. Different patterns of expression are observed for each of the three IE RNAs during permissive infection. Comparison of nucleotide sequences of UL36-38 IE cDNAs with those of the genomic sequence verified the use of polyadenylation and splice signals. An additional abundant early class RNA, totally encoded within the UL36-38 region, was found to be expressed from a different promoter than the IE RNAs. In vitro transcription and translation of the IE cDNAs were used to express the UL36-38 IE proteins. These studies define the structure of the UL36-38 gene products and demonstrate that expression from the region is both qualitatively and temporally complex.

Base Sequence↗

PubMed1991

Differentiation of the U937 macrophage cell line removes an early block of HSV-1 infection.

In the human macrophage-like cell line U937, which is resistant to infection with herpes simplex virus type 1 (HSV-1), it was previously shown that resistance can be overcome by inducing differentiation of the cells by treatment with phorbol 12-myristate 13-acetate (PMA). The present data show that differentiation, and not PMA treatment alone, enabled HSV-1 replication, because vitamin D3 and mezerein were also able to cause U937 cells to differentiate to a state permissive for HSV-1 infection. Additionally, a portion of the undifferentiated cells underwent a productive infection when treated with PMA 2 days after infection, suggesting persistence of HSV-1 in these cells. The nonpermissiveness of the undifferentiated cells was further defined. Resistance did not involve differences in virus uptake, because the amounts of viral DNA in the infected cells and nuclei of differentiated and undifferentiated U937 cells were not significantly different early after infection. However, only very low levels of RNA for HSV-1 immediate-early, early, and late genes could be detected in the undifferentiated U937 cells by Northern blot analysis compared with the differentiated U937 cells. These data suggest that the primary block in HSV-1 replication in undifferentiated U937 cells occurred after transport of the viral DNA to the cell nucleus but prior to steady-state accumulation of viral RNA for immediate-early genes.

Cell Differentiation↗

PubMed1991

Human cytomegalovirus UL36-38 and US3 immediate-early genes: temporally regulated expression of nuclear, cytoplasmic, and polysome-associated transcripts during infection.

During permissive in vitro infection, the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) regions give rise to multiple distinct species of RNA in a temporally regulated manner. We have compared the temporally regulated expression of the UL36-38 and US3 regions with that of the well-characterized major IE (MIE) region. Northern (RNA) blot hybridizations with antisense RNA probes were used to examine RNA isolated from infected cells at IE, early, and late times after infection and from cells infected in the presence of anisomycin (used to block de novo viral protein synthesis) or in the presence of phosphonoformate (used to block HCMV DNA synthesis). Different US3 region transcripts were expressed in the cytoplasm during the IE and late phases of infection, with kinetics similar to those of the MIE region. In contrast, various cytoplasmic transcripts from the UL36-38 region were expressed during each of the IE, early, and late phases of infection, including some expressed from IE through late times. The levels of steady-state RNA from the US3 and MIE regions were increased dramatically by infection in the presence of anisomycin, predominantly because of an increase in multiply spliced transcripts. Two of the three UL36-38 IE transcripts were largely unaffected by anisomycin and were expressed abundantly throughout infection, but a third, multiply spliced UL36-38 IE transcript was abundant only during infection in the presence of anisomycin. Nuclear, cytoplasmic, and polysome-associated transcripts from the three IE regions were not significantly different qualitatively or quantitatively. These results suggest that posttranscriptional controls at the levels of nuclear retention or polysome exclusion of transcripts are not operative for the IE region genes. Overall, these results indicate common features of expression of US3, MIE, and UL36-38, in addition to distinctive expression of the UL36-38 region during all temporal phases of expression.

Antigens, Viral↗

PubMed1990

RNA analysis and isolation of cDNAs derived from the human cytomegalovirus immediate-early region at 0.24 map units.

We have examined transcription from the human cytomegalovirus immediate-early genes that are located at approximately 0.24 map units of the viral genome. Upon infection of permissive cells, nonoverlapping transcripts of 1.65 and 1.7 kb were abundant 8 h after infection and at lower levels at later times of infection or in the presence of cycloheximide. A transcript of approximately 3.4 kb that spanned the entire region was less abundant than the smaller RNAs at 8 h after infection, but was not decreased in abundance with cycloheximide treatment. These RNA species appear to correspond to the immediate-early transcripts from this region. Polyadenylated RNA was isolated from human cytomegalovirus infected cells 8 h after infection and used to synthesize directly a cDNA library. cDNAs corresponding to the abundant 1.65- and 1.7-kb transcripts of this region were isolated and characterized.

Blotting, Northern↗

PubMed1988

Effect of macrophage activation on resistance of mouse peritoneal macrophages to infection with herpes simplex virus types 1 and 2.

To define the effect of heterogeneity of murine peritoneal macrophages (M phi) on intrinsic resistance to herpes simplex virus (HSV) infection, several M phi populations were characterized for their response to infection with HSV type 1 (HSV-1) and HSV-2. Steady-state resident M phi (Res M phi) were compared in parallel with M phi activated with Corynebacterium parvum (now designated Propionibacterium acnes) (CP M phi) and thioglycollate-elicited inflammatory M phi (TG M phi). Res M phi were completely non-permissive for productive virus infection and showed no c.p.e. The intrinsic resistance of CP M phi to HSV infection was similar to that of Res M phi, in that the infection was non-productive for infectious virus, but CP M phi showed marked c.p.e. TG M phi showed semi-permissiveness, with virus yields at least 10-fold higher than those in Res M phi and CP M phi, and marked c.p.e. The three distinct intrinsic response patterns were maintained regardless of whether M phi were derived from CD-1 or B6C3F1 mice, or whether the infecting virus was HSV-1 or HSV-2. To define the level at which M phi restrict HSV replication, immunofluorescence assays for viral antigens and hybridization analyses for viral DNA were performed. All M phi populations showed immediate early and early virus polypeptides. Res M phi and CP M phi showed no viral DNA replication, but TG M phi showed moderate levels of viral DNA synthesis that paralleled the infectious virus titres produced. Investigation of the mechanism for the heterogeneous intrinsic antiviral response among the M phi revealed that interferon was not involved, because antiserum to mouse alpha/beta interferon did not alter the intrinsic resistance patterns. Induction of c.p.e. in M phi required live, replication-competent HSV. The involvement of tumour necrosis factor (TNF) in c.p.e. was found to be unlikely; no significant amounts of TNF were detected in the culture medium of the M phi, and inclusion of anti-TNF antibody did not inhibit c.p.e.

Animals↗

PubMed1987

Effects of differentiation of human macrophage-like U937 cells on intrinsic resistance to herpes simplex virus type 1.

We have characterized the effect of differentiation on the resistance of the mononuclear phagocyte to herpes simplex virus type 1 (HSV-1) by using the human mononuclear phagocyte cell line, U937. The replication of HSV-1 was compared in undifferentiated U937 cells and U937 cells induced to undergo differentiation. Undifferentiated U937 cells were highly resistant to HSV-1 infection. Infectious virus levels declined rapidly to less than 0.1 plaque-forming units (PFU) per cell by 24 hr postinfection and the cells were completely resistant to HSV-1-induced cytopathic effect. Differentiation of U937 cells by treatment with phorbol 12-myristate 13-acetate (PMA) was accompanied by a decrease in the resistance to HSV-1 infection. Infectious virus yields were increased greater than 75-fold at 24 hr postinfection, as compared with the undifferentiated U937 cells. PMA-differentiated U937 cells also acquired full susceptibility to HSV-1-induced cytopathic effect. Infectious center assay revealed that the percent of productively infected cells increased from approximately 3% in undifferentiated U937 to greater than 50% in PMA-differentiated cells. U937 cells were also induced to differentiate by treatment with all-trans-retinoic acid, dimethyl sulfoxide, and lymphokine as shown by differentiation-associated changes in morphology and cytochemical enzymes. These cells, however, failed to display increased permissiveness for HSV-1, indicating that the change in permissiveness was uniquely associated with PMA treatment. Undifferentiated U937 cells adsorbed as much virus as PMA-differentiated cells, but immunofluorescence assays, as well as DNA hybridization analysis demonstrated that an early block in HSV-1 replication occurred in undifferentiated U937 cells, before synthesis of viral protein and DNA. PMA-induced differentiation of U937 cells appears to release an early block in HSV-1 replication that is present in undifferentiated U937 cells.

Cell Differentiation↗