Signaling pathways, the ultimate targets of which are nuclear transcription factors, determine the cell's ability to respond to external stimuli. Transduced signals can be interpreted as mitogenic-proliferative, differentiating, or apoptotic, depending on the cell type and the nature and duration of the stimulus. The mitogenic Ras/MEK/MAPK pathway is initiated by growth factor mediated activation of cognate receptors on the cell surface causing the membrane bound G protein Ras to adopt an active, GTP-bound state. Ras coordinates the activation of a cascade of serine (Ser)-threonine (Thr) kinases that begins with Raf and is followed by MAP kinase kinase 1 and 2 (MEK1/2) and mitogen-activated protein kinase (MAPK1/2) and culminates in the expression of the transcription factor c-Fos (41, 46, 48, 67) which is important for promoting cell cycle progression into S phase (14, 39, 44). The GTPase-activating protein Ras-GAP, a major negative regulator of Ras activity, acts to enhance the weak intrinsic Ras GTPase activity by accelerating the hydrolysis rate of bound GTP to GDP (7,27,31,48,57). Ras-GAP inactivation, for example by phosphorylation on Ser-Thr residues, has been implicated in Ras activation (11,24,66,89). The specificity of the signal transduction is determined by protein domains such as SH2, SH3, and PH that bind unique motifs in target proteins for recruitment into signaling complexes (55, 63).Viruses depend on cells for their replication. They take advantage of preexisting signaling pathways or activate them through various strategies, including activation of the RAS/ MEK/MAPK pathway by Ras-GAP inactivation (32). Vaccinia virus (42), simian virus 40 (SV40) (77), human immunodeficiency virus (HIV) type 1 (35), herpesvirus saimiri (38), and coxsackievirus B3 (32) depend upon the activated RAS/MEK/ MAPK pathway for growth. Herpes simplex virus type 1 (HSV-1) increases the levels of transcription factors c-Jun (37) and 23), and its replication is enhanced by activation of the c-Jun N-terminal kinase (JNK) and p38 of the stress-activated signal pathway (58,88). By contrast, HSV-2 increases c-Fos transcription (26), suggesting that the two HSV serotypes use different strategies to take advantage of signaling pathways. However, the mechanism responsible for c-Fos induction in HSV-2-infected cells, the contribution of the Ras/ MEK/MAPK pathway, and their relationship to virus replication are still unknown.The large subunits of HSV-1 and HSV-2 ribonucleotide reductase (RR1) differ from their counterparts in eukaryotic and prokaryotic cells and in other viruses in that they have an intrinsic PK activity (2,5,12,15,17,49,50,60,65). The RR1 promoter has an octamer-TAATGARAT sequence that responds to the VP16-Oct1 complex (18,78,86,87). RR1 is expressed with apparently biphasic kinetics that consist of immediate-early (IE; also known as ␣) and early components (3,30,84,86,87,90). Expression is independent of the regulatory IE protein ICP4 (18,78,86,87). AP-1 cognate sites in the HSV-2 RR1 (also known as ICP10) promo...

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The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for β-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

Hepatitis A virus (HAV) is a major cause of infectious hepatitis in humans. In this respect, bivalve mollusks pose a major health concern because they are filter feeders and can concentrate the virus up to 900-fold from contaminated water. Detection of HAV has been hampered because wild-type HAV grows poorly if at all in cell culture. Here we describe a technique for the detection of HAV in shellfish based on reverse transcription coupled with the polymerase chain reaction. RNA is isolated from hard-shell clam tissue and reverse transcribed with avian myeloblastosis virus reverse transcriptase. A portion of the cDNA pool is then amplified with primers specific for HAV. In experiments with an in vitro-synthesized HAV transcript, we were able to detect HAV sequence in the presence of a 200-million-fold excess of shellfish RNA. When intact virus was added to shellfish tissue before the isolation of RNA, the method was capable of detecting 10 viral RNA molecules in a reaction mixture.