A large number of temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1) in the gene encoding the immediate-early transcriptional regulatory protein, ICP4, have been isolated and characterized with respect to expression of the immediate-early, early, and late viral gene products. The hallmark of these mutants is the overproduction of immediate-early gene products and the underproduction of early and late gene products. The present study involves the preliminary genetic and molecular characterization of two unique regulatory mutants of HSV-1, ts48 and ts303. Genetically, both mutants exhibit inefficient complementation with eight ts mutants in complementation group 1-2, which defines the gene for ICP4, and marker rescue experiments place the mutations in both mutants in the 3' portion of the coding sequence for ICP4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of ts48-and ts303-infected cell polypeptides synthesized at the nonpermissive temperature demonstrates that immediate-early polypeptides ICP4 and ICP27 are overproduced, with the simultaneous production of early polypeptides ICP6, ICP8, gB, and others. Immediate-early polypeptides are resynthesized upon temperature shift-up early in infection; however, shift-up late in infection does not result in the resynthesis of immediate-early polypeptides. Late gene products are either absent or underrepresented under long-term labeling conditions. To examine the effects of the mutations in ts48, ts303, and other ICP4 mutants specifically on early gene expression, trans-induction experiments were performed in cells transfected with the gene for chloramphenicol acetyltransferase under early gene control (tk) and superinfected with KOS, tsB32, ts48, and ts303. Mutant tsB32 did not induce chloramphenicol acetyltransferase activity above the basal level; however, ts48 and ts303 induced chloramphenicol acetyltransferase activity nearly equal to wild-type levels. Fifteen to fifty percent of wild-type levels of viral DNA are synthesized at the nonpermissive temperature in ts48and ts303-infected cells, indicating that immediate-early and early gene functions are intact (or nearly so) and that the block in ts48 and ts303 is in a regulatory event subsequent to that exhibited by other mutants in complementation group 1-2 which are DNA-. mutants induced nearly wild-type levels of early polypeptides and moderate levels of viral DNA at the nonpermissive 767

Summary The aciduricity of Streptococcus mutans is an important virulence factor of the organism, required to both out-compete commensal oral microorganisms and cause dental caries. In this study, we monitored transcriptional changes that occurred as a continuous culture of either an acid-tolerant strain (UA159) or an acid-sensitive strain (fabM::Erm) moved from steady-state growth at neutral pH, experienced glucose-shock and acidification of the culture, and transitioned to steady-state growth at low pH. Thus, the timing of elements of the acid tolerance response (ATR) could be observed and categorized as acute vs. adaptive ATR mechanisms. Modulation of BCAA biosynthesis, DNA/protein repair mechanisms, ROS metabolizers, and PTS occurred in the initial acute phase, immediately following glucose-shock, while up-regulation of F1F0-ATPase did not occur until the adaptive phase, after steady-state growth had been re-established. In addition to the archetypal ATR pathways mentioned above, glucose-shock led to differential expression of genes suggesting a re-routing of resources away from synthesis of fatty acids and proteins, and towards synthesis of purines, pyrimidines and amino acids. These adjustments were largely transient, as upon establishment of steady-state growth at acidic pH, transcripts returned to basal expression levels. During growth at steady-state pH 7, fabM::Erm had a transcriptional profile analogous to that of UA159 during glucose-shock, indicating that even during growth in rich media at neutral pH, the cells were stressed. These results, coupled with a recently established collection of deletion strains (Quivey et al., 2015), provide a starting point for elucidation of the acid tolerance response in S. mutans.

Pneumocystis carinii is an important pulmonary pathogen responsible for morbidity and mortality in patients with AIDS. The acute-phase response (APR), the primary mechanism used by the body to restore homeostasis following infection, is characterized by increased levels of circulating fibrinogen (FBG). Although the liver is the primary site of increased FBG synthesis during the APR, we unexpectedly discovered that FBG is synthesized and secreted by lung alveolar epithelial cells in vitro during an inflammatory stimulus. Therefore, we sought to determine whether lung epithelial cells produce FBG in vivo using animal models of P. cariniipneumonia (PCP). Inflammation was noted by an influx of macrophages to P. carinii-infected alveoli. Northern hybridization revealed that γ-FBG mRNA increased two- to fivefold in P. carinii-infected lung tissue, while RNA in situ hybridization demonstrated increased levels of γ-FBG mRNA in the lung epithelium. Immunoelectron microscopy detected lung epithelial cell-specific production of FBG, suggesting induction of a localized inflammatory response resembling the APR. A systemic APR was confirmed by a two- to fivefold upregulation of the levels of hepatic γ-FBG mRNA in animals with PCP, resulting in a corresponding increase in levels of FBG in plasma. Furthermore, immunoelectron microscopy revealed the presence of FBG at the junction of cell membranes of trophic forms of P. carinii organisms aggregated along the alveolar epithelium. These results implicate FBG in the pathogenesis of PCP in a manner similar to that of the adhesive glycoproteins fibronectin and vitronectin, which are known to participate in intra-alveolar aggregation of organisms and adherence ofP. carinii to the lung epithelium.

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