Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution.

Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.     

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.     

Borna disease virus, a negative-strand RNA virus, transcribes in the nucleus of infected cells.

Borna disease virus, an unclassified infectious agent, causes immune-mediated neurologic disease in a wide variety of animal hosts and may be involved in pathogenesis of selected neuropsychiatric diseases in man. Initial reports suggested that Borna disease virus is a single-stranded RNA virus. We describe here a method for isolation of viral particles that has allowed definitive identification of the genome as containing a negative-polarity RNA. Further, we show that the viral mRNAs are transcribed in the nucleus.     

Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors

Effective gene therapy depends on the efficient transfer of therapeutic genes and their protein products to target cells. Lentiviral vectors appear promising for virus-mediated gene delivery and long-term expression in nondividing cells. The herpes simplex virus type 1 tegument protein VP22 has recently been shown to mediate intercellular transport of proteins, raising the possibility that it may be helpful in a setting where the global delivery of therapeutic proteins is desired. To investigate the effectiveness of lentiviral vectors to deliver genes encoding proteins fused to VP22, and to test whether the system is sufficiently potent to allow protein delivery from transduced cells in vitro and in vivo, fusion constructs of VP22 and the enhanced green fluorescent protein (EGFP) were prepared and delivered into target cells by using HIV-1-based lentiviral vectors. To follow the spread of VP22-EGFP to other cells, transduced COS-7 cells were coplated with a number of different cell types, including brain choroid plexus cells, human endothelial cells, H9 cells, and HeLa cells. We found that VP22-EGFP fusion proteins were transported from transduced cells to recipient cells and that such fusion proteins accumulated in the nucleus and in the cytoplasm of such cells. To determine the ability to deliver fusion proteins in vivo, we injected transduced H9 cells as well as the viral vector directly into the brain of mice. We present evidence that VP22-EGFP fusion proteins were transported effectively from lentivirus transduced cells in vivo. We also show that the VP22-EGFP fusion protein encoded by the lentivirus is transported between cells. Our data indicate that such fusion proteins are present in the nucleus and in the cytoplasm of neighboring cells. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22.     

Analysis of viral DNA sequences in hamster cells transformed by herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) DNA was treated with four restriction endonucleases (EcoRI, HincIII, Bgl II, and Xba I) and eight fragments were purified and labeled with 32P in vitro. The kinetics of renaturation of each of the fragments was measured in the presence of DNA extracted from 333-8-9, a hamster cell line transformed by UV light-inactivated HSV-2 strain 333, and from a series of cloned derivatives and their tumor lines. All of the lines examined contained a partial set of viral sequences present at only a few copies per cell. Passage of the cell lines in tissue culture or in animals resulted in partial loss of viral DNA. Two blocks of sequences were present in most of the lines examined; those mapping at positions 21--33 of the HSV-2 genome were detected in seven of seven cell lines tested and those at positions 60--65 were detected in six of eight. Other sequences from the L component can also be present in the DNA of HSV-2-transformed hamster cells.     

Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammlian cells.

The survival of UV-irradiated herpes simplex virus on UV-irradiated Vero cells was increased over that on unirradiated cells. A time period between irradiation of the host cells and infection with virus was needed to achieve maximum reactivation. In parallel experiments in which the frequencies of occurrence of the forward mutation in the thymidine kinase gene of the virus were measured, growth of herpes simplex virus on UV-irradiated cells yielded progeny virus that had higher frequencies of TK- mutants than did progeny from infections of control cells. The time course of development of this mutagenic effect was the same as that for the development of the UV-reactivation capacity. Furthermore, development of the UV reactivation could be blocked by inhibition of protein synthesis. These results suggest that an "error prone" inducible UV-reactivation phenomenon exists in mammalian cells.     

Pathogenesis of herpes simplex labialis. I. Replication of herpes simplex virus in cultures of epidermal cells from subjects with frequent recurrences

Primary cultures were established with epidermal cells from the skin of 11 patients with frequent episodes of herpes simplex labialis and 13 control subjects with titers of neutralizing antibody to herpes simplex virus (HSV) type 1 but no history of herpetic disease. Confluent monolayers were exposed to HSV type 1 strain E115, and the infection was monitored by assay of the rate of virus appearance in the culture medium. The mean slope of the virus growth curves ([log10 pfu/ml]/log10 hr) was 9.0 in cultures from patients vs. 9.5 in cultures from controls, and the respective mean titers of virus 53 hr after infection were 10(6.8) and 10(6.5) (differences not statistically significant). Genetically controlled host factors may play some role in the clinical response to HSV infection, but variation in the susceptibility of epidermal cells, the natural target for HSV, is not one of the critical determinations.     

Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode.

In situ hybridization histochemistry and immunocytochemistry were used to study the prenatal expression of luteinizing hormone-releasing hormone (LHRH) cells in the mouse. Cells expressing LHRH mRNA and peptide product were first detected on embryonic day 11.5 (E11.5) in the olfactory pit. On E12.5, the majority of LHRH cells were located on "tracks" extending from the olfactory pit to the base of the telencephalon. From E12.5 to E15.5, LHRH cells were detected in a rostral-to-caudal gradient in forebrain areas. Prior to E12.5, cells expressing LHRH mRNA were not detected in forebrain areas known to contain LHRH cells in postnatal animals. Quantitation of cells expressing LHRH mRNA showed that the number of labeled cells on E12.5 (approximately 800) equaled the number of LHRH cells in postnatal animals, but more than 90% of these cells were located in nasal regions. Between E12.5 and E15.5, the location of LHRH cells shifted. The number of LHRH cells in the forebrain increased, while the number of LHRH cells in nasal regions decreased over this same period. These findings establish that cells first found in the olfactory pit and thereafter in forebrain areas express the LHRH gene and correspond to the position of LHRH immunopositive cells found at these developmental times. To further examine the ontogeny of the LHRH system, immunocytochemistry in combination with [3H]thymidine autoradiography was used to determine when LHRH cells left the mitotic cycle. We show that LHRH neurons exhibit a discrete time of birth, suggesting that they arise as a single neuronal population between E10.0 and E11.0. Postnatal LHRH neurons were "birth-dated" shortly after differentiation of the olfactory placode and before LHRH mRNA was expressed in cells in the olfactory pit. Taken together, these studies support the hypothesis that all LHRH cells in the central nervous system arise from a discrete group of progenitor cells in the olfactory placode and that a subpopulation of these cells migrate into forebrain areas where they subsequently establish an adult-like distribution.     

Net -1 frameshifting on a noncanonical sequence in a herpes simplex virus drug-resistant mutant is stimulated by nonstop mRNA

Ribosomal frameshifting entails slippage of the translational machinery during elongation. Frameshifting permits expression of more than one polypeptide from an otherwise monocistronic mRNA, and can restore expression of polypeptides in the face of frameshift mutations. A common mutation conferring acyclovir resistance in patients with herpes simplex virus disease deletes one cytosine from a run of six cytosines (C-chord) in the viral thymidine kinase (tk) gene. However, this mutation does not abolish TK activity, which is important for pathogenicity. To investigate how this mutant retains TK activity, we engineered and analyzed viruses expressing epitope-tagged TK. We found that the mutant's TK activity can be accounted for by low levels of full-length TK polypeptide produced by net -1 frameshifting during translation. The efficiency of frameshifting was relatively high, 3-5%, as the polypeptide from the reading frame generated by the deletion, which lacks stop codons (nonstop), was poorly expressed mainly because of inefficient protein synthesis. Stop codons introduced into this reading frame greatly increased its expression, but greatly decreased the level of full-length TK, indicating that frameshifting is strongly stimulated by a new mechanism, nonstop mRNA, which we hypothesize involves stalling of ribosomes on the polyA tail. Mutational studies indicated that frameshifting occurs on or near the C-chord, a region lacking a canonical slippery sequence. Nonstop stimulation of frameshifting also occurred when the C-chord was replaced with a canonical slippery sequence from HIV. This mechanism thus permits biologically and clinically relevant TK synthesis, and may occur more generally.     

Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis.

When extracellular magnesium is reduced by a factor of 50 (from 1.0 to 0.02 mM), the total intracellular magnesium of a spontaneously transformed clone of 3T3 cells decreases by 30-50%. Protein synthesis rates in these cells were measured as the intracellular magnesium decreased. Protein synthesis rates and magnesium content were found to decrease in parallel with each other. At 3 hr, a decrease to 84% of control values of magnesium content was accompanied by a decrease to 85% of control values of leucine incorporation rates. A larger inhibition had occurred by 12 hr, when the magnesium had decreased to 67% and leucine incorporation rates had decreased to 57%. When magnesium was restored to magnesium-deprived cells, both magnesium content and leucine incorporation increased about 2-fold by 1 hr. In the experiments reported here, initial small changes in magnesium content are associated with changes in protein synthesis rates. This strongly suggests that magnesium is present at a regulatory rather than excess concentration for protein synthesis. The results are consistent with a role for intracellular magnesium in the regulation of protein synthesis and support the hypothesis that magnesium has a central role in the regulation of metabolism and growth.     

A previously unrecognized influenza B virus glycoprotein from a bicistronic mRNA that also encodes the viral neuraminidase.

RNA segment 6 of the influenza B virus genome codes for a previously unidentified polypeptide designated NB. The reading frame for this polypeptide begins with the first AUG codon on the mRNA and overlaps the reading frame for the viral neuraminidase by 292 nucleotides. The amino acid sequence of polypeptide NB deduced from the nucleotide sequence of the B/Lee/40 strain consists of 100 amino acids with a molecular weight of 11,242. The sequence contains four potential glycosylation sites, and the protein has been found to be glycosylated in infected cells. NB has not been found in virions. Sucrose gradient sedimentation and analysis of the structure of the mRNA by nuclease S1 mapping and sequence analysis by the primer extension method indicated that polypeptide NB and the neuraminidase are translated from a single bicistronic mRNA. A protein analogous to NB has not been found with influenza A virus, and this represents a major difference between the two virus types.     

Long-term increases in neurotransmitter release from neuronal cells expressing a constitutively active adenylate cyclase from a herpes simplex virus type 1 vector.

Signal-transduction pathways mediate a wide range of short-term changes in the physiology of neuronal systems from invertebrates to mammals. However, examples of long-term changes in neuronal physiology mediated by these pathways have been limited to invertebrate systems. In this report, long-term changes in the physiology of mammalian neurons were studied by using genetic intervention to cause a long-lasting activation of the cAMP pathway. The catalytic domain of yeast adenylate cyclase (cyr), encoding a constitutive enzyme activity, was expressed in neuronal cells infected with a defective herpes simplex virus vector (pHSVcyr). In PC-12 cells infected with pHSVcyr, increases were seen in cAMP levels, protein kinase A activity, protein phosphorylation, phosphorylation of the tyrosine hydroxylase protein kinase A site (Ser40), and catecholamine release. Infection of sympathetic neurons with pHSVcyr increased cAMP levels, protein phosphorylation, and catecholamine release. Yeast adenylate cyclase immunoreactivity and elevated cAMP levels were localized to the cell bodies of sympathetic neurons. The increase in neurotransmitter release was both Ca(2+)- and activity-dependent and persisted for at least 1 week after infection of the sympathetic neurons, suggesting that sustained physiological activation of the cAMP pathway may mediate long-term changes in the neuronal physiology of mammalian systems.     

Aerosol vaccination with a sendai virus temperature-sensitive mutant (HVJ-pB) derived from persistently infected cells.

Experimental infections of mice with a Sendai virus temperature-sensitive (ts) mutant (HVJ-pB) were studied. Infection with the ts mutant induced the priming effect of interferon production and both humoral and cellular immune responses, although the ts mutant virus neither multiplied satisfactorily in the respiratory tracts of mice nor caused appreciable histopathologic lesions. Inoculation with the ts mutant protected mice from subsequent challenge with a parental wild-type virus. The efficacy of this protection began as little as 1 day after vaccination and continued for at least 12 weeks. It is suggested that serum antibodies were efficacious in the nasal turbinates, while specific immune spleen cells act more protectively in the lungs.     

Translation of Rous sarcoma virus RNA in a cell-free system from ascites Krebs II cells.

The template activities of the 60-70S RNA complex and of the 30-40S subunit RNA species of Rous sarcoma virus were tested in a cell-free protein-synthesizing system from mouse ascites Krebs II cells. Stimulation of protein synthesis over the endogenous background was about 2-fold with 30-40S viral RNA and about 1.3-fold with 60-70S viral RNA as template. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the predominant polypeptide synthesized in vitro in response to 30-40S RNA of Rous sarcoma virus had a molecular weight of 75,000-80,000. This polypeptide could be precipitated by antiserum against the group-specific antigens of the virus, although its molecular weight is higher than that of virion group-specific antigen proteins. Analysis of tryptic digests of the protein made in vitro indicates similarity to tryptic digests from authentic virion group-specific proteins. It is concluded that part of the RNA from Rous sarcoma virus is translated in vitro into a high-molecular-weight protein, perhaps a precursor of the virion group-specific proteins.     

Biosynthesis of beta-endorphin from beta-lipotropin and a larger molecular weight precursor in rat pars intermedia.

When isolated rat pars intermedia cells were incubated for 10 min with radioactive amino acids, one major labeled protein with a molecular weight of 30,000 +/- 1500 was extracted. This protein was shown to contain in its sequence the antigenic determinants for corticotropin and beta-melanotropin by immunoprecipitation. When the radioactivity incorporated into this large molecular weight protein during the first 10 min was chased by a further incubation in presence of an excess of unlabeled amino acid, the initial protein was degraded into several smaller peptides including beta-endorphin and beta-lipotropin. Another 18,000-dalton peptide was also observed and was tentatively identified as a large molecular form of corticotropin. From the kinetics of the maturation of the initial precursor, it is concluded that the initial cleavage of the 30,000-dalton peptide gives rise to beta-lipotropin and the 18,000-dalton form of corticotropin. beta-Lipotropin is subsequently cleaved to form beta-endorphin.