single strand DNA while RecG shows a 3Ј to 5Ј DNA Department of Molecular Microbiology, Research Institute for helicase activity (Tsaneva et al., 1993; Whitby et al., Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 1994). Osaka 565, Japan In the RuvABC pathway, RuvC is a specific endonucle-1 Corresponding author ase that resolves Holliday junctions (Dunderdale et al., 1991;Iwasaki et al., 1991). RecG, which functions in The RecG protein of Escherichia coli is a DNA helicase the alternative pathway, has been suggested to promote that promotes branch migration of the Holliday juncjunction resolution by driving branch migration in the tions. We found that overproduction of RecG protein opposite direction from that promoted by RecA protein drastically decreased copy numbers of ColE1-type (Whitby et al., 1993;Whitby and Lloyd, 1995). These plasmids, which require R-loop formation between the authors proposed that RecG plays a role in preventing template DNA and a primer RNA transcript (RNA unproductive recombination by reversing the invasion of II) for the initiation of replication. RecG efficiently a 5Ј single-stranded DNA end into the homologous duplex inhibited in vitro ColE1 DNA synthesis in a reconstitu- . ted system containing RNA polymerase, RNase HI andThe recG and ruv genes play different roles in alternative DNA polymerase I. RecG promoted dissociation of mechanisms of stable DNA replication (SDR), which do RNA II from the R-loop in a manner that required not require the chromosomal replication origin (oriC), ATP hydrolysis. These results suggest that overDnaA initiator protein or concomitant protein synthesis produced RecG inhibits the initiation of replication (Asai and Kogoma, 1994a). Mutations in the ruv or recG by prematurely resolving the R-loops formed at the gene stimulate inducible SDR (iSDR), which is a part of replication origin region of these plasmids with its SOS responses, suggesting that this mode of SDR involves unique helicase activity. The possibility that RecG D-loops or Holliday junctions made by RecA (Asai and regulates the initiation of a unique mode of DNA Kogoma, 1994b). recG mutants, but not ruv mutants, replication, oriC-independent constitutive stable DNA stimulate constitutive SDR (cSDR), which was found replication, by its activity in resolving R-loops is disoriginally in RNase HI-defective (rnhA) mutants, and recG cussed.rnhA double mutants are inviable. These results suggested

Background: All the ruvA, ruvB and ruvC mutants of Escherichia coli are sensitive to treatments that damage DNA, and are mildly defective in homologous recombination. It has been reported that the ruv mutants form nonseptate, multinuclear filaments after low doses of UV irradiation, dependent on the sfiA gene product. In vitro, the RuvAB complex promotes the branch migration of Holliday junctions, and RuvC resolves the junctions endonucleolytically.

The products of the recG and ruvAB genes of Escherichia coli are both thought to promote branch migration of Holliday recombination intermediates by their junction specific helicase activities in homologous recombination and recombination repair. To investigate the in vivo role of the recG gene, we examined the effects of a recG null mutation on cell division and chromosome partition. After UV irradiation at a low dose (5J/ m 2 ), ∆recG mutant formed filamentous cells with unpartitioned chromosomes. A mutation in the sfiA gene, which encodes an SOS-inducible inhibitor of septum formation, partially suppressed filamentation of recG mutant cells, but did not prevent the formation of anucleate cells. The sensitivity to UV light and the cytological phenotypes after UV irradiation of a recA recG double mutant were similar to a recA single mutant, consistent with the role of recG, which is assigned to a later stage in recombination repair than recA. The recG ruvAB and recG ruvC double mutants were more sensitive to UV, almost as sensitive as the recA mutant and showed more extreme phenotypes concerning filamentation and chromosome nondisjunction, both after UV irradiation and without UV irradiation than either recG or ruv single mutants. The recG polA12 (Ts) mutant, which is temperature sensitive in growth, formed filamentous cells with centrally located chromosome aggregates when grown at nonpermissive temperature similar to the UV irradiated recG mutant. These results support the notion that RecG is involved in processing Holliday intermediates in recombination repair in vivo. We suggest that the defect in the processing in the recG mutant results in accumulation of nonpartitioned chromosomes, which are linked by Holliday junctions.

Human RSV causes an annual epidemic of respiratory tract illness in infants and in elderly. Mechanisms by which RSV antagonizes IFN-mediated antiviral responses include inhibition of type I IFN mRNA transcription and blocking signal transduction of JAK/STAT family members. The suppressor of cytokines signaling (SOCS) gene family utilizes a feedback loop to inhibit cytokine responses and block the activation of the JAK/STAT signaling pathway. To evaluate the potential of SOCS molecules to subvert the innate immune response to RSV infection, eight SOCS family genes were examined. RSV infection up-regulated SOCS1, SOCS3, and CIS mRNA expression in HEp-2 cells. Suppression of SOCS1, SOCS3 and CIS by short interfering ribonucleic acid (siRNA) inhibited viral replication. Furthermore, inhibition of SOCS1, SOCS3, or CIS activated type I IFN signaling by inducing STAT1/2 phosphorylation. These results suggest that RSV infection escapes the innate antiviral response by inducing SOCS1, SOCS3 or CIS expression in epithelial cells.

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As the number of patients treated with acyclovir (ACV) has increased, increasing numbers of ACV-resistant (ACV r ) herpes simplex virus (HSV) and varicella-zoster virus strains have been isolated, mainly from immunocompromised patients (8). The selectivity of ACV as an antiherpesvirus drug is based on its specific interaction with virus-encoded enzymes, thymidine kinase (TK) and DNA polymerase (DNA Pol). Therefore, the mechanisms responsible for ACV resistance are mutations in the TK and/or DNA Pol polypeptides (1). A previous largescale clinical study on ACV r HSV strains isolated from patients infected with human immunodeficiency virus indicated that 96% of ACV r HSV mutants were low producers of, or deficient in, TK activity (TK Ϫ ), with 4% being TK mutants with an altered substrate specificity. No DNA Pol mutants were isolated (12).It is generally believed that ACV r strains arise from naturally occurring mutations during DNA replication and are selected by ACV both in vitro (cell culture experiments) and in vivo (patients) (2). This hypothesis was formed from the following observations: (i) without exposure to ACV, approximately 0.3 to 20 ACV r mutants occur in 10 4 PFU of clinical HSV-1 isolates (11,19,26,28) and of a laboratory strain grown from one plaque (2); (ii) ACV r isolates under ACV selective pressure represent only a small proportion of the viable virions in the original virus population (19); and (iii) no ACV mutagenic activity has been detected in previous studies (3, 30).However, there is still a possibility that ACV does influence the development of ACV r strains during ACV treatment. To address the question of whether or not ACV induces mutation, we chose penciclovir (PCV), which is similar to ACV both in structure and in its need for phosphorylation by virus TK for its anti-HSV action, as a control drug and isolated a series of ACV r and PCV-resistant (PCV r ) strains emerging during serial passages of HSV-1 in the presence of ACV or PCV. Sequencing of the TK and DNA Pol genes showed differential mutation patterns in the ACV r and PCV r isolates, suggesting that one, if not both, of the drugs had a differential effect on the generation of drug-resistant mutants. MATERIALS AND METHODS Cells and viruses.A human osteosarcoma TK-deficient cell line, 143B/ TK Ϫ neo R , was kindly supplied by Riken Cell Bank, Tsukuba, Japan. Human embryo lung (HEL) fibroblasts, Vero, and 143B/TK Ϫ neo R cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with 10% calf serum. The VR-3 strain of HSV-1 described previously (6) was obtained from the American Type Culture Collection, Rockville, Md. This virus strain was plaque purified three times, grown in HEL cells for less than three passages, and stored in small portions at Ϫ80°C.Isolation of drug-resistant viruses. ACV r and PCV r HSV-1 strains were isolated by serial passage of the reference VR-3 strain in the presence of increasing concentrations of ACV or PCV, as follows. Twenty PFU of the VR-3 strain were inoculated onto HEL cell mon...

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Nedd4 is a family of ubiquitin E3 ligases that regulate numerous cellular processes. In this report, we showed that alpha- and beta-herpesviruses have membrane proteins that regulate the function of the Nedd4 family members. Although the homology search score was quite low, UL56 of herpes simplex virus type 1 and 2, ORF0 of varicella-zoster virus, UL42 of human cytomegalovirus, and U24 of human herpesvirus 6A, 6B, and 7 all possess at least one PPxY (PY) motif in their cytoplasmic domain, and are able to bind with Itch, a member of the Nedd4 family. These viral proteins altered the localization of Itch and decreased Itch expression in co-expressing cells. In addition, these viral proteins reduced the production of retrovirus vectors through the regulation of the Nedd4 family of proteins. U24, but not the other proteins, effectively reduced CD3ε expression on the T cell surface. These viral molecules are thought to contribute to the specific function of each virus through the regulation of Nedd4 family activity.