Use of differential display reverse transcription-PCR to reveal cellular changes during stimuli that result in herpes simplex virus type 1 reactivation from latency: upregulation of immediate-early cellular response genes TIS7, interferon, and interferon regulatory factor-1.

The detailed mechanism which governs the choice between herpes simplex virus (HSV) latency and reactivation remains to be elucidated. It is probable that altered expression of cellular factors in sensory neurons leads to induction of HSV gene expression resulting in reactivation. As an approach to identify novel cellular genes which are activated or repressed by stimuli that reactivate HSV from latency and hence may play a role in viral reactivation, RNA from explanted trigeminal ganglia (TG) was analyzed by differential display reverse transcription-PCR (DDRT-PCR). Nearly 50 cDNAs whose mRNA level was modified by the stress of explantation were isolated and sequenced. We present a listing of a spectrum of altered RNAs, including both known and unknown sequences. Five of those differentially displayed transcripts were identified as interferon-related murine TIS7 mRNA. These results were confirmed in both infected and uninfected ganglia by quantitative RNase protection assay and immunostaining. Alpha and beta interferons and interferon regulatory factor-1 (IRF-1) were also induced by explantation. In addition, we have identified sequences that correspond to IRF-1 consensus binding sites in both HSV type 1 origins of replication. Our findings suggest that physiological pathways that include these cellular factors may be involved in modulating HSV reactivation.

Gene expression during reactivation of herpes simplex virus type 1 from latency in the peripheral nervous system is different from that during lytic infection of tissue cultures.

Herpes simplex virus (HSV) replicates in peripheral tissues and forms latent infections in neurons of the peripheral nervous system. It can be reactivated from latency by various stimuli to cause recurrent disease. During lytic infection in tissue culture cells, there is a well-described temporal pattern of (i) immediate-early, (ii) early, and (iii) late gene expression. However, latency is characterized by little if any expression of genes of the lytic cycle of infection. During reactivation, the pattern of gene expression is presumed to be similar to that during the lytic cycle in tissue culture, though recent work of W. P. Halford et al. (J. Virol. 70:5051-5060, 1996) and P. F. Nichol et al. (J. Virol. 70:5476-5486, 1996) suggests that it is modified in neuronal cell cultures. We have used the mouse trigeminal ganglion explant model and reverse transcription-PCR to determine the pattern of viral and cellular gene expression during reactivation. Surprisingly, the pattern of viral gene expression during lytic infection of cell cultures is not seen during reactivation. During reactivation, early viral transcripts were detected before immediate-early transcripts. The possibility that a cellular factor upregulates early genes during the initial reactivation stimulus is discussed.

Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex.

A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication. We report here the characterization of three such mutants: TusP42S, TusE49K, and TusH50Y. TusP42S and TusE49K arrest DNA replication in vivo at 36% of the efficiency of wild-type Tus, whereas TusH50Y functions at 78% efficiency. The loss of replication arrest activity did not correlate with changes in the stability of the Tus-TerB complexes formed by the mutant proteins. TusE49K formed a more stable protein-DNA complex than wild-type Tus (t1/2 of 178 versus 149 min, respectively) and TusP42 had a 9-min half-life, yet these two mutants showed identical efficiencies for replication arrest. When tested in vitro using a helicase assay or an oriC replication system, we observed a general, but imperfect, correlation between the in vivo and in vitro assays. Finally, the half-lives of the mutant protein-DNA complexes suggested that the domain of Tus where these mutations are located is positioned close to the DNA in the Tus-Ter complex.

Isolation and characterization of mutants of Tus, the replication arrest protein of Escherichia coli.

Mutations in the tus gene of Escherichia coli, which encodes the replication arrest protein Tus, were isolated using a selection scheme based on the plasmid pHV750T2+, which transforms tus mutants at a much higher frequency than wild type strains. Seven mutants containing single nucleotide substitutions were isolated, and all of these mutants showed reduced or complete loss of DNA binding and replication arrest activity. Two of the mutant proteins, containing a valine (A173V) or threonine (A173T) in place of the alanine normally found at amino acid 173, were purified and characterized further. A173T had a 4100-fold lower affinity for Ter sites than wild type Tus and was unable to halt DNA replication in vivo or inhibit DnaB-catalyzed strand displacement in an in vitro helicase assay. A173V showed a 130-fold lower affinity for Ter sites than wild type Tus but was still able to arrest DNA replication in vivo, suggesting that protein-protein interactions were responsible for Tus-mediated arrest of DNA replication. In addition, we found that A173V was a weak inhibitor of DnaB-catalyzed strand displacement in vitro, yet halted DNA replication in vivo at 75% of the efficiency of wild type Tus. We concluded from these observations that the standard in vitro helicase assay was inadequate for measuring Tus activity.

Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli.

Tus, a DNA-binding protein, mediates arrest of DNA replication in Escherichia coli. Tus binds to DNA sequences called Ter sites, located in the terminus region of the chromosome, and forms replication-arrest complexes that block movement of DNA replication forks in a polar fashion. We have analyzed Tus to determine some of its physical parameters and biochemical characteristics. Native Tus had an 8(20,w) of 3.2, a Stokes' radius of 23 A, an axial ratio of 2, and a molar absorption coefficient of 39,700 M-1 cm-1. The data also indicated that Tus existed as a monomeric protein in solution and when complexed with its cognate DNA binding site. Secondary structure estimated from the circular dichroism spectrum suggested that Tus consisted of 40% alpha-helix, 0% beta-sheet, 15% turn, and 45% aperiodic structure. The isoelectric point of native Tus (pH 7.5) was significantly different than that calculated from its amino acid sequence (pH 10.1), possibly because the tertiary structure of Tus perturbs the ionization of several residues. In addition, partial proteolytic digests of free Tus protein did not produce a subfragment of Tus that retained DNA binding activity, but did demonstrate that Tus was resistant to proteolysis when complexed with a Ter site.