Herpes simplex virus 1 (HSV-1) encodes an endoribonuclease that is responsible for the shutoff of host protein synthesis [virion host shutoff (VHS)-RNase]. The VHS-RNase released into cells during infection targets differentially four classes of mRNAs. Thus, (a) VHS-RNase degrades stable cellular mRNAs and α (immediate early) viral mRNAs; (b) it stabilizes host stress response mRNAs after deadenylation and subsequent cleavage near the adenylate-uridylate (AU)-rich elements; (c) it does not effectively degrade viral β or γ mRNAs; and (d) it selectively spares from degradation a small number of cellular mRNAs. Current evidence suggests that several viral and at least one host protein (tristetraprolin) regulate its activity. Thus, virion protein (VP) 16 and VP22 neutralize the RNase activity at late times after infection. By binding to AU-rich elements via its interaction with tristetraprolin, the RNase deadenylates and cleaves the mRNAs in proximity to the AU-rich elements. In this report we show that another virion protein, UL47, brought into the cell during infection, attenuates the VHS-RNase activity with respect to stable host and viral α mRNAs and effectively blocks the degradation of β and γ mRNAs, but it has no effect on the processing of AU-rich mRNAs. The properties of UL47 suggest that it, along with the α protein infected cell protein 27, attenuates degradation of mRNAs by the VHS-RNase through interaction with the enzyme in polyribosomes. Mutants lacking both VHS-RNase and UL47 overexpress α genes and delay the expression of β and γ genes, suggesting that overexpression of α genes inhibits the downstream expression of early and late genes.

ABSTRACT A herpes simplex virus tegument protein brought into the cell during infection and designated the virion host shutoff protein (VHS) is an endoribonuclease that degrades mRNA. The prevailing view for many years has been that the VHS-RNase does not discriminate between cellular and viral RNAs and that the viruses prevail because the accumulation of viral transcripts outpaces their degradation. Here we report the following. (i) The degradation of viral mRNA made during infection of Vero or HEp-2 cells proceeds at a much-reduced rate compared to that of cellular mRNA. In effect, viral mRNAs are largely stable, whereas cellular mRNAs are rapidly degraded or, in the case of AU-rich mRNA, cleaved and rendered dysfunctional. (ii) In contrast to viral mRNAs made after infection, viral mRNAs expressed by plasmids transfected into cells prior to infection are degraded after infection at a rate comparable to that of cellular mRNAs. Moreover, the mRNA encoded by the transfected plasmid is hyperadenylated in the infected cell. Hyperadenylation but not degradation of mRNAs is blocked by actinomycin D. The results indicate that VHS-mRNA discriminates between viral and cellular mRNA but only in the context of infection and that discrimination is not based on the sequence of the mRNA but most likely on one or more viral factors expressed in the infected cell.

During diabetes, structural and functional changes in the alimentary tract are known to take place resulting in an increased absorption of intestinal glucose and alterations in the activities of brush-border disaccharidases. To elucidate the effect of administrating polysaccharide fromGynura divaricata(PGD) on disaccharidase activities, the specific activities of intestinal disaccharidases, namely sucrase, maltase and lactase, were measured in streptozotocin-induced diabetic rats. Normal control and diabetic rats were treated by oral administration with PGD. Specific activities of intestinal disaccharidases were increased significantly during diabetes, and amelioration of the activities of sucrase and maltase during diabetes was clearly visible by the treatment with PGD. However, the increased activity of lactase during diabetes mellitus was remarkably alleviated by the administration of PGD only in the duodenum. Meanwhile, oral sucrose tolerance tests demonstrated that PGD alleviated the hyperglycaemia during diabetes mellitus, resulting from the amelioration in the activities of intestinal disaccharidases. The present investigation suggests that PGD exerted an anti-diabetic effect partly via inhibiting the increased intestinal disaccharidase activities of diabetic rats. This beneficial influence of administration of PGD on intestinal disaccharidases clearly indicates their helpful role in the management of diabetes.

The virion host shutoff (VHS) RNase tegument protein released into cells by infecting virus has two effects. Preexisting stable mRNAs (e. g., GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) are rapidly degraded. Stress response RNAs containing AU-rich elements (AREs) in the 3' untranslated region (3'UTR) are deadenylated and cleaved, but the cleavage products persist for hours, in contrast to the short half-lives of ARE-containing mRNAs in uninfected cells. At late times, the VHS RNase is neutralized by the viral structural proteins VP16 and VP22. A recent study (J. A. Corcoran, W. L. Hsu, and J. R. Smiley, J. Virol. 80: 9720-9729, 2006) reported that, at relatively late times after infection, ARE RNAs are rapidly degraded in cells infected with Delta ICP27 mutant virus and concluded that ICP27 "stabilizes" ARE mRNAs. We report the following. (i) The rates of degradation of ARE mRNA at early times (3 h) after infection with the wild type or the Delta ICP27 mutant virus are virtually identical, and hence ICP27 plays no role in this process. (ii) In noncomplementing cells, VHS RNase or VP22 is not synthesized. Therefore, the only VHS that is active is brought into cells by the Delta ICP27 mutant. (ii) The VHS RNase brought into the cells by the Delta ICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in Delta ICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in Delta VHS RNase mutant-virus-infected cells and does not by itself support the hypothesis that ICP27 stabilizes ARE mRNAs. (iii) Concurrently, we present the first evidence that VHS RNase interacts with ICP27 most likely when bound to cap-and poly(A)-binding proteins, respectively.

The virion host shutoff protein product of the U L 41 gene of herpes simplex virus 1 is an endoribonuclease that selectively degrades mRNAs during the first hours after infection. Specifically, in contrast to the events in uninfected cells or cells infected with a mutant lacking the RNase, in wild-type virus-infected cells mRNA of housekeeping genes exemplified by GAPDH is degraded rapidly, whereas mRNAs containing AU elements are cleaved and the 5′ cleavage product of these RNAs persists for many hours. We report that in wild-type virus-infected cells there was a rapid increase in the number and size of processing bodies (P-bodies). These P-bodies were also preset in cycloheximide (CHX)-treated cells but not in either treated or untreated uninfected cells or cells infected with the RNase minus mutant. Additional studies revealed that polyribosomes extracted from cytoplasm of wild-type virus-infected cells treated with CHX and displayed in sucrose gradients contained ribosome-loaded, truncated AU-rich mRNAs lacking the 3′ UTR and poly(A) tails. The results suggest that the virion RNase is bound to polyribosomes by virtue of the reported association with translation machinery and cleaves the RNAs 5′ to the AU elements. In contrast to the slow degradation of the of the residual 5′ domain, the 3′ UTR of the AU-rich mRNA and the GAPDH mRNA are rapidly degraded in wild-type virus-infected cells.

The virion host shutoff (vhs) protein encoded by the U L 41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (γ) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the U L 41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

The herpes simplex virus 1 ORF U(L)41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears rapidly, a large 5' domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3' untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.

ABSTRACT Earlier we reported that NF-κB is activated by protein kinase R (PKR) in herpes simplex virus 1-infected cells. Here we report that in PKR −/− cells the yields of wild-type virus are 10-fold higher than in PKR +/+ cells. In cells lacking NF-κB p50 ( nfkb1 ), p65 ( relA ), or both p50 and p65, the yields of virus were reduced 10-fold. Neither wild-type nor mutant cells undergo apoptosis following infection with wild-type virus. Whereas PKR +/+ and NF-κB +/+ control cell lines undergo apoptosis induced by the d120 (Δα4) mutant of HSV-1, the mutant PKR −/− and NF-κB −/− cell lines were resistant. The evidence suggests that the stress-induced apoptosis resulting from d120 infection requires activation of NF-κB and that this proapoptotic pathway is blocked in cells in which NF-κB is not activated or absent. Activation of NF-κB in the course of viral infection may have dual roles of attempting to curtain viral replication by rendering the cell susceptible to apoptosis induced by the virus and by inducing the synthesis of proteins that enhance viral replication.

Microarray data reported elsewhere indicated that herpes simplex virus 1 induces the up-regulation of nuclear factor κB (NF-κB)-regulated genes, including that of its inhibitor, IκBα, consistent with the reports that wild-type virus induces the activation of NF-κB. In this report we show that activation of NF-κB in infected cells is linked to the activation of protein kinase R (PKR). Specifically: ( i ) PKR is activated in infected cells although the effects of the activated enzyme on protein synthesis are negated by the viral gene γ 1 34.5, which encodes a protein phosphatase 1α accessory factor that enables the dephosphorylation of the α subunit of eukaryotic translation initiation factor 2. NF-κB is activated in wild-type murine embryonic fibroblasts but not in related PKR-null cells. ( ii ) In cells infected with a replication-competent Δγ 1 34.5 mutant (R5104), but carrying a U S 11 gene expressed early in infection, eukaryotic translation initiation factor 2α is not phosphorylated, and in in vitro assays, PKR bound to the U S 11 protein is not phosphorylated on subsequent addition of double-stranded RNA. Here we report that this mutant does not activate PKR, has no effect on the accumulation of IκBα, and does not cause the translocation of NF-κB in infected cells. ( iii ) One hypothesis advanced for the activation of NF-κB is that it blocks apoptosis induced by viral gene products. The replication-competent R5104 mutant does not induce the programmed cell's death. We conclude that in herpes simplex virus 1-infected cells, activation of NF-κB depends on activation of PKR and that NF-κB is not required to block apoptosis in productively infected cells.

The accumulation of cellular transcripts from cells infected with herpes simplex virus 1 (HSV-1) as measured with the aid of Affymetrix microchips has been reported elsewhere. Among these transcripts were genes that respond to stress and that could have a noxious effect on viral replication. We have selected the stress-inducible cellular gene encoding the immediate-early response protein IEX-1 to verify and determine the significance of the accumulation of these transcripts in infected cells. We report that we verified the increase in accumulation of IEX-1 transcripts after infection by Northern analyses and real-time PCR. These transcripts reach peak levels between 3 and 7 h after infection and decrease thereafter. However, IEX-1 protein was detected in cells 1 h after infection but not at later intervals. Studies designed to elucidate the failure of IEX-1 protein to be synthesized revealed the following points. (i) IEX-1 RNA transported to the cytoplasm after 1 h of infection consisted of at least two populations, a partially degraded population and a population consisting of unspliced IEX-1 RNA. Neither of these RNAs could translate the authentic IEX-1 protein. (ii) The partially degraded IEX-1 RNA was not detected in the cytoplasm of cells infected with a mutant virus lacking the U(L)41 gene encoding the virion host shutoff protein (vhs). Although degradation of RNA mediated by vhs was reported to be 5' to 3', the partially degraded IEX-1 RNA lacked the 3' sequences rather than the 5' sequences. (iii) The unspliced pre-RNA form containing the IEX-1 intron sequences was detected in the cytoplasm of cell infected with wild-type virus but not in those infected with a mutant lacking the alpha27 gene encoding the infected cell protein No. 27. (iv) Overexpression of IEX-1 protein by transduction of the gene prior to infection with 1 PFU of HSV-1 per cell had no effect on the accumulation of late genes and virus yield. We conclude that the failure of IEX-1 to express its protein reflects the numerous mechanisms by which the virus thwarts the cells from expressing its genes after infection.

Religion, which mainly means Buddhism in China, should be one of the dominant indications of Chinese cultural regionazation. The study on Chinese Buddhistical geography began in the late 1920s. Up to date, the related study included two types : one was the studies in Buddhistcal history, and another was the specialized studies. The former was just a supplementary method in history studies, so its achievements can only be found oddly in history books. It was the later type that dealt with the problems of geography. The achievements were in two aspects : one was to discuss the import route at a level of international communication, and the other was to research the geographical distribution of eminent monks and temples. The author assumes that: 1) It is essentially to pay attention to historical changes. And this should be a distinguishing feature of Chinese cultural geography. 2) Buddhism is composed of several layers. For the study of Buddhistical Geography, it could be devided into three layers: faithful layer for popular people; religious layer for Buddhists; and academic layer for eminent monks. The study should be based on the knowing of the layered-structure of Buddhism and the analyzing of the factors of each layers, and then we can come to an all-round conclusion. 3) The study methods should be innovated.