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T M Block

Publications and source records attributed to T M Block.

At least 37 records · Page 2Linked to original sources
PubMed1996

Correlation between herpes simplex virus type 1 rate of reactivation from latent infection and the number of infected neurons in trigeminal ganglia.

The presence of wild-type herpes simplex virus type 1 (HSV-1) and several latency associated transcript (LAT) region mutants within the trigeminal ganglia (TG) of latently infected mice was examined. A combination of methods including conventional in situ hybridization to detect viral LAT and an in situ DNA polymerase chain reaction (PCR) to detect viral DNA was used. These data show that, for all virus strains in which a comparison was possible, the population of neurons expressing detectable levels of LAT was approximately one-third the total number of viral DNA-containing cells. In addition, in situ PCR analysis revealed that mutants such as 17 delta Sty. 17 delta BstE, and 17 delta S/N, which contain deletions within the LAT locus which do not affect the kinetics of viral reactivation from explanted murine TG, are present in as many neurons as wild-type virus. This was true regardless of the ability to induce accumulation of intact 2.0-kb LAT. On the other hand, mutant 17 delta N/H, which contains a deletion removing the LAT promoter and surrounding genomic region and reactivates slowly from explanted TG, was present in only one-sixth as many neurons as wild-type virus. These data show that detection of mutants unable to synthesize or accumulate 2.0-kb LAT (such as 17 delta N/H) is possible with in situ DNA PCR and that the slow reactivation phenotype of 17 delta N/H correlates with a reduced number of HSV DNA-containing neurons.

Animals↗
PubMed1996

In vivo epinephrine reactivation of ocular herpes simplex virus type 1 in the rabbit is correlated to a 370-base-pair region located between the promoter and the 5' end of the 2.0 kilobase latency-associated transcript.

A rabbit ocular model of epinephrine-induced herpes simplex virus type 1 reactivation was employed to study the effect of a deletion in the latency-associated transcript domain. A viral construct derived from 17Syn+, designated 17deltaSty, has a deletion of 370 nucleotides between genomic positions 118880 and 119250. 17deltaSty has been shown to reactivate with wild-type virus kinetics from explants of trigeminal ganglia from latently infected mice. To determine the behavior of this mutant in an in vivo, inducible reactivation system, rabbit corneas were infected with 17Syn+, 17deltaSty, or its rescuant, 17detlaSty-Res. After viral latency was established, transcorneal epinephrine iontophoresis was performed. The rabbits latently infected with 17deltaSty exhibited a significantly reduced ability to undergo adrenergically induced reactivation, i.e., viral shedding in the tears, compared with rabbits infected with either 17Syn+ or 17deltaSty-Res. However, quantitative PCR demonstrated similar numbers of viral genomes in the trigeminal ganglia from rabbits latently infected with all three viruses, and all three viruses reactivated in vitro with wild-type kinetics in an explant cocultivation assay. These studies indicate that the 370-bp region deleted in the 17deltaSty construct plays a role in epinephrine-induced reactivation.

Animals↗
PubMed1996

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.

1-Deoxynojirimycin↗
PubMed1996

Evidence that two latency-associated transcripts of herpes simplex virus type 1 are nonlinear.

The latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) are the only viral gene products that accumulate to abundant levels in latently infected cells. Others have reported species of 2.0, 1.50, and 1.45 kb; only the 2.0-kb species is seen in productively infected cells, and there is evidence that it behaves as an intron. We examined the LATs both in trigeminal ganglia of latently infected mice and in productively infected cultures of monkey CV-1 cells. After glyoxalation, RNA was subjected to high-resolution agarose gel electrophoresis and Northern (RNA) analysis, a procedure capable of resolving linear and nonlinear RNA species. Under these conditions, we resolved the 2.0-kb LAT into two species; the slower species was much more abundant and had a mobility significantly slower than expected for a linear RNA. To test the hypothesis that this RNA was in fact nonlinear, we used partial hydrolysis by sodium carbonate and oligonucleotide-directed RNase H digestion. These procedures changed the mobility of the slower species into that of the faster species. Similarly, the mobility of the 1.50-kb LAT, which was much more abundant than the 1.45-kb LAT, was changed by these procedures to that of the 1.45-kb LAT. Our data show that the two major LAT species are nonlinear, and they support an interpretation of stable lariat structures.

Animals↗
PubMed1996

A herpes simplex virus type 1 mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics.

The latency associated transcripts (LATs) are the only abundant viral gene products detected during latent herpes simplex virus (HSV) infection of peripheral nerves in animals and people. A LAT promoter has been identified and mutant viruses with lesions removing the promoter and surrounding region have been observed to reactivate slowly from trigeminal ganglia (TG) explanted from latently infected mice. Previous work has shown that most mutants with lesions limited to regions downstream of the LAT promoter reactivate normally. Therefore, to help map the boundaries of the slow reactivation phenotype, a mutant virus with lesions located immediately upstream of the LAT promoter was constructed and called 17 delta S/N. 17 delta S/N contains a 437 nucleotide (nt) deletion 332 nts upstream of the TATAA box of the LAT promoter. In productively infected cells, 17 delta S/N failed to synthesize detectable amounts of the 1.1 and 1.8 kb transcripts which are produced during wild-type infections and are specified by a region just upstream of the LAT promoter. However, 17 delta S/N did produce normal amounts of LAT in tissue culture as well as in neurons derived from latently infected cells, as ascertained by Northern blot and in situ hybridization analysis. Moreover, in latently infected mice, 17 delta S/N established and maintained infection in as many neurons as did wild type virus, as determined by in situ polymerase chain reaction (PCR) to detect viral DNA. Finally, the virus reactivated from TG derived from latently infected mice with kinetics indistinguishable from those of wild-type virus. Therefore, reactivation from latency, in this model system, does not appear to require function from the viral genomic region located immediately upstream of the LAT promoter.

Animals↗
PubMed1995

In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal ganglia of latently infected mice.

The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected by an in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with LAT probes. Sensitivity assays were shown to detect a single copy cellular gene in 48% of neuronal cell bodies. The results suggest that in situ PCR is an effective method to locate and detect HSV-1 within latently infected neurons. Moreover, the number of neurons found to be harboring HSV-1, by the method of in situ PCR, which does not depend upon virus gene expression, is within threefold of the number detected by in situ hybridization for LAT. Therefore, this report describes the first detection of HSV-1 DNA in latently infected murine trigeminal ganglia by the method of indirect in situ PCR, and compares the findings to the number of neurons expressing LAT, as assessed by conventional in situ hybridization.

Animals↗
PubMed1994

Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor of the oligosaccharide-trimming enzyme alpha-glucosidase I. Hepatitis B virus (HBV) contains three surface proteins (HBs proteins) of different sizes that are singly or doubly N-glycosylated and are essential for the formation of infectious virus. Therefore, the replication and secretion of HBV in the human hepatoma cell line HepG2 were studied in the presence of NBDNJ. In the stably HBV-transfected HepG 2.2.15 cells and in HBV-infected HepG2 cells, NBDNJ suppressed secretion of HBV particles and caused intracellular retention of HBV DNA. The secretion of subviral particles was less affected. These data suggest that inhibitors of oligosaccharide trimming may be useful for antiviral therapy of hepatitis B and for the study of the intracellular transport of the viral glycoproteins.

1-Deoxynojirimycin↗
PubMed1994

Analysis of a herpes simplex virus type 1 LAT mutant with a deletion between the putative promoter and the 5' end of the 2.0-kilobase transcript.

A herpes simplex virus type 1 strain 17 mutant with a deletion between genomic nucleotides 118880 and 119250 was constructed and called 17 delta Sty. The deletion removes most of a putative secondary LAT promoter (called LAPII) as well as 370 of the first 449 nucleotides of the proposed 8.5-kb transcript believed to be the precursor of 2.0-kb LAT. 17 delta Sty was shown to produce major 2.0-kb LATs in tissue culture. Moreover, trigeminal nerves from latently infected mice contained an intact 1.45- to 2.0-kb LAT as well as the minor LATs which are recognized by probes specific for regions downstream of the 2.0-kb LAT. Finally, 17 delta Sty reactivated with normal kinetics from the trigeminal ganglia of latently infected mice in the explant cocultivation assay and egressed from tissue culture cells as efficiently as wild-type virus. These results clearly show that the region deleted in 17 delta Sty is dispensable for intact 2-kb LAT production, viral egress in tissue culture, and normal reactivation from latently infected neurons in mice.

Animals↗
PubMed1993

An HSV LAT null mutant reactivates slowly from latent infection and makes small plaques on CV-1 monolayers.

A Herpes simplex virus type I (HSV-I) strain 17 mutant deleted between the NotI and HpaI restriction sites of the latency associated transcript (LAT) region has been constructed. The mutant, therefore, contains a deletion of the putative LAT promoter and is called 17N/H. The 17N/H isolate established latent infections in mice nearly as efficiently as its wildtype parent. However, like other LAT null mutants, 17N/H reactivates from explanted ganglia with much slower kinetics than its LAT competent parent. In tissue culture, although 17N/H produces as much virus per cell as its strain 17 parent, it produces small plaques. The small plaque phenotype appears to be due to the inability of the virus to be released from the infected cell into the medium, following low but not high multiplicities of infection (m.o.i.). The mutant was also shown to produce an aberrant LAT homologous transcript of 1.1 kb as well as overproduce an approximately 29,000-Da HSV-specific polypeptide, which is barely detectable in wildtype infected cells. Rescuants of the 17N/H defect were constructed using a 10-kb restriction fragment containing viral sequences spanning the deletion, make large plaques, and have reactivation patterns and infected cell gene product profiles indistinguishable from the 17 parent. This shows that the phenotypes observed in 17N/H are reversed when the deletion, or at most sequences within 5 kb of each side of the deletion, is corrected. The possibilities that the defect in viral egress from infected cell, the small LAT homologous transcript, and the accumulation of the 29,000 Da polypeptide are related to the delayed reactivation kinetics are discussed.

Base Sequence↗
PubMed1993

Latency and reactivation of Marek's disease virus in B lymphocytes transformed by avian leukosis virus.

The physical and biological state of the Marek's disease virus (MDV) genome in avian leukosis virus (ALV)-transformed cells is characterized using cell lines established from ALV tumours co-infected with the SB-1 strain of MDV. The MDV genome within the ALV-transformed cells was found to be methylated at 5' CpG 3' dinucleotides. Less than 2% of the tumour cells expressed MDV antigen and only one virus plaque that was characteristic of an MDV infection was noted when tumour cells were cocultured with fibroblasts permissive for a productive MDV infection. However, when methylation of the MDV genome was prevented by culturing the tumour cell lines in the presence of 5-azacytidine, both MDV antigen expression and viral replication increased. Based on these results, it appears that MDV resides within the ALV-transformed cells in a latent state and that MDV latency might be influenced, to some extent, by methylation of the MDV genome.

Alpharetrovirus↗
PubMed1992

Genetically engineered bacteria to identify and produce anti-viral agents.

We have prepared a strain of Escherichia coli that expresses both the HIV protease and a Tet protein which has been modified to contain the HIV protease recognition sequence. When the protease is expressed, the bacteria will not grow in the presence of tetracycline. However, when the protease is inhibited the bacteria can grow in tetracycline containing media (Block and Grafstrom 1990). We have selected spontaneously arising Tet resistant mutants and have screened them for those that could be producing an inhibitor of HIV protease. The problems in the construction of this strain and the characterization of the various Tetr mutants are discussed.

Antiviral Agents↗
PubMed1992

Persistence of Marek's disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell.

Animals↗
PubMed1990

Novel bacteriological assay for detection of potential antiviral agents.

A prototype assay for the initial screening of potential antiviral agents that uses bacterial growth on selective media is described. The human immunodeficiency virus (HIV) protease recognition sequence was inserted into the tetracycline resistance (Tet) protein encoded by plasmid pBR322 of Escherichia coli. Expression of both the HIV protease and the modified Tet protein prevented growth in the presence of tetracycline. However, inhibition of the HIV protease restored tetracycline resistance. Thus, potential HIV protease inhibitors can be identified by their ability to confer tetracycline resistance to this bacterial strain. The assay is simple, rapid, and inexpensive, and this concept can be applied to the search for inhibitors of other viral proteases.

Antiviral Agents↗
PubMed1990

A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) accumulate in neuronal nuclei of latently infected ganglia. Explant reactivation kinetics of LAT deletion mutants in the mouse eye model have suggested a role for the LATs in the reactivation process. This report describes the construction and characterization of an HSV-1 strain HFEM mutant, TB1, disrupted within both copies of the LAT gene. TB1 contains a 440-base-pair segment of bacteriophage lambda DNA in place of a 168-base-pair deletion within the transcribed portion of the LAT gene. The 2.0-kilobase LAT was not produced after infection of tissue culture cells with TB1, but a 0.7- to 0.8-kilobase RNA was expressed. TB1 did establish latent infection after corneal inoculation as efficiently as the parental virus, and its reactivation kinetics from explanted ganglia were similar to those of HFEM. During latent infection with TB1, HSV-1 transcripts were not detectable. Rescuant virus (TB1-R) contained intact LAT genes, synthesized full-length LAT transcripts during productive infection in tissue culture, and reactivated from ganglionic explants of latently infected mice with normal kinetics. Thus, any function these transcripts have in the reactivation process appears to include the region between the putative LAT promoter and the disruption in TB1--a region of approximately 1,600 nucleotides, 800 of which encode the LATs.

Animals↗
PubMed1988

The effect of photoperiod on diurnal rhythms of serum gonadotrophins, prolactin and melatonin in ovariectomized heifers.

Fourteen heifers, ovariectomized prepubertally, were used in two experiments to test the hypotheses that (1) there are diurnal patterns in circulating concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin and melatonin and (2) that these diurnal patterns would be altered by changing photoperiod. In experiment 1 (Exp. 1) animals were randomly assigned to either increasing photoperiod (I) (n = 7) or decreasing photoperiod (D) (n = 7) for a period of 8 weeks. In the second experiment (Exp. 2) four heifers from each photoperiod group were assigned to the opposite treatment for another 4 weeks forming four subgroups; continued increasing photoperiod (II) (n = 3), increasing followed by decreasing photoperiod (ID) (n = 4), decreasing followed by increasing photoperiod (DI) (n = 4) and continued decreasing photoperiod (DD) (n = 3). At weeks 4, 8 (Exp. 1) and 12 (Exp. 2) the animals were cannulated and blood samples were taken hourly from 1030 hr to 0830 hr (23 samples). In Exp. 1, animals exposed to either I or D did not differ in circulating concentrations of LH, FSH, prolactin and melatonin. Concentrations of LH and melatonin but not FSH or prolactin were higher in animals during the scotophase than the photophase. In Exp. 2 LH, FSH and prolactin, but not melatonin concentrations were higher in animals exposed to I (II and ID) than D (DI and DD). Animals exposed to I had higher circulating concentrations of FSH and animals exposed to both I and D had higher concentrations of melatonin during the scotophase. Neither LH nor prolactin concentrations differed between scotophase and photophase. The abrupt switch from one photoperiod treatment to the other did not significantly affect LH, FSH or prolactin but did alter the patterns and concentrations of circulating melatonin. Animals that remained in the same photoperiod treatment had similar melatonin concentrations; but animals changed from I to D had higher serum melatonin concentrations than those changed from D to I. These data support the hypothesis that there are diurnal changes in circulating concentrations of melatonin in the prepubertal bovine female, with concentrations higher during scotophase than photophase. In addition, under certain photoperiodic conditions there were diurnal rhythms in gonadotropins. These data also support the hypothesis that changing photoperiod alters the diurnal pattern in circulating melatonin concentrations.

Animals↗
PubMed1987

Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants.

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.

Base Sequence↗