Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.

The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.

Quiescent viral genomes in human fibroblasts after infection with herpes simplex virus type 1 Vmw65 mutants.

The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent 'latent' state. Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i. In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of beta-galactosidase was not detected after infection of IFN-alpha-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells. A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus. The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form. A small number of remaining linear genomes resulted from incomplete uncoating of input virus.

Myc induces cyclin D1 expression in the absence of de novo protein synthesis and links mitogen-stimulated signal transduction to the cell cycle.

Mitogen-activated signal transduction frequently leads to the induction of the c-myc proto-oncogene, but the subsequent molecular events downstream of Myc protein expression which promote cell cycle progression remain unclear. To study Myc-specific effects, without the complexity of the broader proliferative response evoked by serum, we employed the MycER-inducible system in the non-transformed Rat-1 cell line. We demonstrate that activation of wild-type, but not mutant, MycER is sufficient to transiently induce cyclin D1 RNA as well as protein expression to physiological levels, and promote G0/G1 to S phase transition of the cell cycle. Stimulation of endogenous cyclin D1 RNA is rapid and clearly evident within 30 min of MycER-activation, reaching a peak at 3 h. Nuclear run-on analysis demonstrates that this induction occurs at the transcriptional level with a fivefold increase in the rate of transcription. Moreover, MycER induces cyclin D1 transcription with equal efficacy in the presence or absence of de novo protein synthesis. Our work shows that Myc and cyclin D1 lie consecutively in a major proliferation-control pathway, and together create a pivotal connection between signal transduction and cell cycle control.

Herpes simplex virus immediate early gene expression in the absence of transinduction by Vmw65 varies during the cell cycle.

The requirement for the herpes simplex virus type 1 (HSV-1) protein Vmw65 (VP16) for activation of immediate early (IE) gene expression was examined in synchronized HeLa cells. Analyses of IE RNA levels were conducted during infection with a viral Vmw65 mutant, in1814. The results revealed an increased requirement for Vmw65 when cultures reached G2 phase of the cell cycle. The levels of IE RNAs 1, 2, and 4 were reduced 5-10 times more in G2 than G1/S for in1814-infected cells when compared to cells infected with wild-type virus or 1814R (a rescued virus), and similar but smaller effects were observed on IE RNA 3 levels. The relative decrease at G2 was reversed by resynchronization of cells to G1/S. Mutant in1814 formed plaques less efficiently on cells at G2 than on cells synchronized at G1/S. The results show that, in the absence of functional Vmw65, HSV-1 IE gene expression and replication vary during the cell cycle.

Hexamethylene bisacetamide stimulates herpes simplex virus immediate early gene expression in the absence of trans-induction by Vmw65.

Hexamethylene bisacetamide (HMBA) and DMSO are known to induce differentiation of cultured erythroleukaemic cells and to enhance the reactivation of latent herpes simplex virus (HSV) after explantation of ganglia. We report that the presence of these compounds in cell culture medium overcomes the replication defect of in1814, an HSV-1 mutant with an insertion mutation that inactivates the virion trans-inducing factor, Vmw65 (VP16). The effect of HMBA was not cell type-specific and was attained even by a short exposure (1.5 to 5 h) to the agent early after infection. The presence of HMBA resulted in an increase in immediate early (IE) RNA accumulation after infection of cells in the presence of cycloheximide, such that RNA levels in in1814-infected cells approached the values observed in wild-type HSV-1-infected cells in the absence of HMBA. Transport of viral DNA to the cell nucleus was not affected by HMBA. The results suggest that HMBA- and DMSO-mediated enhancement of reactivation from latency is due to an increase in IE RNA production. In addition, these studies demonstrate a primary effect of HMBA on gene regulation which may be a paradigm for initial events during erythroleukaemic cell differentiation.

Further characterization of ts1-8, a mutant of herpes simplex virus type 1.

Our initial characterization of a herpes simplex virus type 1, temperature sensitive host shutoff mutant, called ts1-8, revealed that it has a low plaquing efficiency and exhibits a defect in the shutoff of host polypeptide synthesis and host DNA replication at the nonpermissive temperature of 39.5 degrees C. Using intratypic marker rescue experiments the ts plaquing mutation was mapped to a 557 bp region. Sequence analysis and complementation studies revealed that the low plaquing efficiency phenotype is due to a mutation in the glycoprotein B gene converting Pro-357 to Ser. This novel tsgB mutation is located in a conserved region of gB and it is distinct from the delayed host shutoff mutation (dhs).

Physical mapping of two herpes simplex virus type 1 host shutoff loci: rescue of each ts mutation occurs with two unique cloned regions of the viral genome.

Two complementing temperature-sensitive (ts) herpes simplex virus type 1 (HSV-1) mutants, PAA1rts1 and ts199, were defective in viral DNA synthesis and in the shutoff of cellular macromolecular synthesis at 39.5 degrees C, the nonpermissive temperature. PAA1sts1 and PAA1rts1+ recombinants and PAA1rts1+ revertants were used to examine the contributions of the PAA1r mutation and the ts1 mutation of PAA1rts1 in affecting the levels of viral and cellular DNA synthesized at 34 and 39.5 degrees C. The results of this study suggests an interaction between the viral DNA polymerase and the ts1+ gene product during HSV-1 DNA replication and possibly in the inhibition of host DNA synthesis by HSV-1. Physical mapping of the ts mutations present in ts199 and the PAA1sts1 recombinant ts1-8 were performed by intratypic marker rescue experiments. Surprisingly, both the ts1-8 and ts199 mutations were rescued by two cloned fragments: ts1-8 by BglII-K (map coordinates 0.095 to 0.163) and BglII-I (map coordinates 0.314 to 0.417), while ts199 was rescued by BglII-K and BglII-O (map coordinates 0.163 to 0.197). In more refined mapping experiments, the regions between coordinates 0.347 to 0.378 and 0.126 to 0.163 were able to rescue the ts1-8 mutation. Southern hybridization analysis confirmed that the fragments that rescued ts1-8 and those that rescued ts199 had homology, as predicted by the physical mapping results.

Temperature-sensitive herpes simplex virus type 1 mutants defective in the shutoff of cellular DNA synthesis and host polypeptide synthesis.

Two temperature-sensitive herpes simplex virus type 1 mutants, ts 1-8 and ts 199, belonging to different complementation groups, were isolated. Both mutants were defective in the shutoff of host DNA synthesis at 39.5 degrees C (nonpermissive temperature). ts 1-8 exhibited intermediate levels of viral DNA synthesis at 39.5 degrees C, while ts 199 was completely deficient in viral DNA synthesis at 39.5 degrees C. Comparative polyacrylamide gel electrophoresis of the ts 1-8, ts 199 and wild-type viral-coded polypeptides and cellular proteins produced in vivo at 34 degrees C and 39.5 degrees C during various periods post infection was performed. The results indicated that ts 1-8 and ts 199 were temperature-sensitive for the secondary suppression of host polypeptide synthesis. Production of the beta (early) and gamma (late) viral polypeptides was slightly delayed in the mutant-infected cells at early times post infection at both 34 degrees C and 39.5 degrees C. This delayed protein production was not evident at later times post-infection. The ts 1-8 and ts 199 mutants were distinct from the HSV-1 viron-associated host shutoff (vhs) mutants of Read and Frenkel (J. Virol. 46 (1983) 498).

Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.