Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.     

Ribonucleotide Reductase Activity of Synchronized KB Cells Infected with Herpes Simplex Virus

The replication of herpes simplex virus (HSV) is unimpeded in KB cells which have been blocked in their capacity to synthesize deoxyribonucleic acid (DNA) by high levels of thymidine (TdR). Studies showed that the presence of excess TdR did not prevent host or viral DNA replication in HSV-infected cells. In fact, more cellular DNA was synthesized in infected TdR-blocked cells than in uninfected TdR-blocked cells. This implies that the event which relieved the TdR block was not specific for viral DNA synthesis but allowed some cellular DNA synthesis to occur. These results suggested that HSV has a means to insure a pool of deoxycytidylate derivatives for DNA replication in the presence of excess TdR. We postulated that a viral-induced ribonucleotide reductase was present in the cell after infection which was not inhibited by thymidine triphosphate (TTP). Accordingly, comparable studies of the ribonucleotide reductase found in infected and uninfected KB cells were made. We established conditions that would permit the study of viral-induced enzymes in logarithmically growing KB cells. A twofold stimulation in reductase activity was observed by 3 hr after HSV-infection. Reductase activity in extracts taken from infected cells was less sensitive to inhibition by exogenous (TTP) than the enzyme activity present in uninfected cells. In fact, the enzyme extracted from infected cells functioned at 60% capacity even in the presence of 2 mm TTP. These results support the idea that a viral-induced ribonucleotide reductase is present after HSV infection of KB cells and that this enzyme is relatively insensitive to inhibition by exogenous TTP.     

Herpes Simplex Virus Types 1 and 2 Completely Help Adenovirus-Associated Virus Replication

In addition to adenoviruses, which are capable of completely helping adenovirus-associated virus (AAV) multiplication, only herpesviruses are known to provide any AAV helper activity, but this activity has been thought to be partial (i.e., AAV DNA, RNA, and protein syntheses are induced, but infectious particles are not assembled). In this study, however, we show that herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are in fact complete AAV helpers and that AAV type 2 (AAV2) infectivity yields can approach those obtained when coinfections are carried out with a helper adenovirus. AAV helper activity was demonstrated in KB cells with two HSV-1 strains (11124 and 17MP) and an HSV-2 strain (HG52). Each herpesvirus supported AAV2 multiplication with comparable efficiency. AAV2 multiplication was similarly efficient in HSV-1 coinfections of HeLa cells, whereas lower yields were obtained in HEp-2 and primary human embryonic kidney cells. HSV-1 also supported AAV1 multiplication in HeLa cells but, at corresponding multiplicities of infection, AAV1 grew less efficiently than AAV2. Comparisons of the time courses of AAV2 DNA, RNA, and protein syntheses after coinfection with either adenovirus type 5 or HSV-1 revealed that, in each case, the onset of synthesis and attainment of maximal synthesis rate occurred earlier in coinfections with HSV-1. These findings demonstrate the linkage of AAV macromolecular synthesis to an event(s) in the helper virus cycle. Aside from this temporal association, helper-related differences in AAV macromolecular synthesis were not apparent.     

Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells I. Sequence of Viral Replication

A comparison, under standardized conditions, of herpes simplex virus (HSV) and human cytomegalovirus (CMV) revealed differences in viral morphology, in the timing of their infectious cycles, and in several morphological events during those cycles. Structural distinctions between the two viruses included the coating of unenveloped cytoplasmic CMV capsids, but not those of HSV, and a variation in the structure of their cores. Since the two cycles were carried out in the same host cell strain under conditions of one-step growth (input multiplicity = 10 PFU/cell), it was possible to construct time scales locating the major events of each cycle. Comparison of the two showed that HSV replicated and released progeny within 8 h postinfection, whereas CMV required 4 days. These results correlated well with those of concurrent plaque assays. Within the longer CMV cycle, most of the major events appeared retarded to a similar degree, and no obvious limiting step in particle production could be identified. Distinctions between the two cycles included the following: condensation of the chromatin in HSV- but not CMV-infected cells; the greater tendency of HSV to produce membrane alterations; and the appearance of cytoplasmic dense bodies in CMV- but not HSV-infected cells. Identification of these differences even under identical conditions of culture and infection strongly implies that they result from intrinsic differences in the nature of the viruses, and are not caused by variations in experimental conditions.     

Stepwise Evolution of the Herpes Simplex Virus Origin Binding Protein and Origin of Replication

The herpes simplex virus replicon consists of cis-acting sequences, oriS and oriL, and the origin binding protein (OBP) encoded by the UL9 gene. Here we identify essential structural features in the initiator protein OBP and the replicator sequence oriS, and we relate the appearance of these motifs to the evolutionary history of the alphaherpesvirus replicon. Our results reveal two conserved sequence elements in herpes simplex virus type 1, OBP; the RVKNL motif, common to and specific for all alphaherpesviruses, is required for DNA binding, and the WP XXXGAXXFXX L motif, found in a subset of alphaherpesviruses, is required for specific binding to the single strand DNA-binding protein ICP8. A 121-amino acid minimal DNA binding domain containing conserved residues is not soluble and does not bind DNA. Additional sequences present 220 amino acids upstream from the RVKNL motif are needed for solubility and function. We also examine the binding sites for OBP in origins of DNA replication and how they are arranged. NMR and DNA melting experiments demonstrate that origin sequences derived from many, but not all, alphaherpesviruses can adopt stable boxI/boxIII hairpin conformations. Our results reveal a stepwise evolutionary history of the herpes simplex virus replicon and suggest that replicon divergence contributed to the formation of major branches of the herpesvirus family.Herpesviruses have been found in animal species ranging from molluscs to man. According to the International Committee on Taxonomy of Viruses, the order of Herpesvirales consists of three families as follows: Alloherpesviridae, Herpesviridae, and Malacoherpesviridae (1). The subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae are found within the family of Herpesviridae. The events leading to establishment of a new virus species are poorly understood, but in the case of herpesviruses it is commonly assumed that viruses co-evolve with their hosts (2). Herpesviruses have thus become well adapted to their hosts and may reside in a latent state in the host for a lifetime with little or no overt signs of infection. Upon reactivation, the infectious virus will be released. The viruses remain faithful to their hosts, and infections across species borders are rare but may under specific circumstances give rise to fatal disease.Replication of herpes simplex virus type 1 (HSV-1),² requires a cis-acting DNA sequence, the replicator, termed oriS or oriL, an initiator protein, OBP or UL9 protein, and a replisome composed of DNA polymerase, helicase-primase, and a single strand DNA-binding protein referred to as ICP8 or UL29 protein (3). OBP assisted by ICP8 can in an ATP-dependent reaction unwind double-stranded oriS (4, 5). The resulting single-stranded DNA adopts a hairpin conformation, which is stably bound by OBP (6, 7). The herpesvirus replisome once assembled on DNA is capable of synthesizing leading and lagging strands processively in a coordinated fashion (8). DNA replication is likely to start on circular molecules produced by the action of DNA ligase IV/XRCC4 and proceed in a θ-type manner (9, 10). Later, a rolling circle mode of replication dominates giving rise to characteristic head-to-tail concatemers. The initiator protein OBP appears to be strictly required only during the first few hours of the infectious cycle (11, 12).The HSV-1 origin binding protein was first isolated using an assay monitoring specific binding to HSV-1 oriS (13). A minimal DNA-binding site was identified using footprinting techniques as well as binding studies with double-stranded oligonucleotides (14). For alphaherpesviruses the high affinity binding site is always TTCGCAC, with a minor exception for CHV1 also referred to as monkey B virus (Table 1).3 The C-terminal 317 amino acids of alphaherpesvirus OBP can be isolated as a soluble protein, which remains capable of high affinity binding to the sequence TTCGCAC (15). The C-terminal domain of HSV-1 OBP, here referred to as ΔOBP, binds as a monomer to the major groove in the DNA and makes contacts with base pairs as well as the deoxyribose-phosphate backbone (16, 17). ΔOBP binds DNA specifically with an estimated K_d of 0.3 nm, a value that is highly influenced by the composition of the assay buffer (16). At high protein concentrations ΔOBP form aggregates, which, still in a sequence-specific manner, binds DNA (16). A number of studies have attempted to define amino acids involved in DNA binding (18–21). In addition, sequence comparisons between alphaherpesviruses and roseoloviruses have helped to identify amino acids in OBP potentially involved in DNA binding as well as corresponding recognition sequences in origins of DNA replication (22–25). However, a comprehensive and quantitative study of evolutionarily conserved amino acids required for DNA binding is still lacking.

TABLE 1

Functionally significant sequence motifs for herpes simplex virus replicon evolutionOrigins of DNA replication have been identified from sequence analysis. For roseoloviruses the sequences for two slightly different binding sites for OBP have been listed (31). The number and orientation of OBP-binding sites in relation to an AT-rich spacer sequence are schematically presented by symbols > and <. Note that since a virus often has more than one origin of replication they may exist as variants. This is indicated by symbols within parentheses. The conserved amino acids within the ICP8-binding motif are shown in boldface type. Sources and nomenclature for DNA sequences have been presented in Footnote 3.Open in a separate windowThe single strand DNA-binding protein, ICP8 encoded by the UL29 gene, is involved in initiation of DNA replication, and it also participates in the elongation phase at the replication fork (4, 5, 26). ICP8 forms a specific complex with OBP (26, 27). Studies with deletion mutants have demonstrated that important sequences are found close to the C terminus of OBP, but amino acids directly participating in the high affinity interaction have not been identified. The interaction is biologically significant, because deletion of the extreme C terminus enhances the helicase activity of OBP but reduces origin-dependent DNA synthesis (26).The HSV-1 oriS contains three copies of the binding site for OBP; two binding sites, box I and box II, are high affinity sites, and the third binding site, box III, has a very low affinity for OBP (14) (Fig. 1). All sites are required for efficient replication, and in a competitive situation there is a strong selection for the most efficient replicator sequence (28). Box III and box I are arranged in a palindrome, which becomes rearranged upon activation to form an alternative conformation, most likely a hairpin (4, 6, 7). Point mutations that prevent formation of a hairpin reduce replication, and compensatory mutations restore complementary base pairing as well as the ability to replicate (7).Open in a separate windowFIGURE 1.Schematic presentation of the herpes simplex virus replicon. Upper part, HSV-1 oriS. The linear genome contains three homologous replication origins, two copies of oriS and one copy oriL, and encodes seven replication proteins. Middle part, HSV-1 OBP. OBP or UL9 protein is a superfamily II DNA helicase as well as a sequence-specific DNA-binding protein. Here the helicase domain is represented by two connected ellipsoids, and the C-terminal DNA binding is drawn as a circle. The OBP-binding sites in oriS are shown. The OBP dimer binds two double-stranded DNA box I oligonucleotides but only one hairpin with a single-stranded tail (48). The figure is intended to demonstrate conformational changes affecting the DNA binding domain, referred to as ΔOBP, during activation of oriS. Lower part, DNA binding domain ΔOBP. A schematic presentation of the following three motifs discussed in this publication: the F553 XX KYL motif required for proper folding of the DNA binding domain, the R756VKNL motif necessary for DNA binding, and the W839PXXXGAXXFXXL motif involved in binding to ICP8.To learn more about the mechanism of virus evolution, we have examined the evolutionary history of some functionally significant features of the HSV-1 replicon and related them to a sequence-based evolutionary tree. The results indicate that replicon divergence, characterized by the stepwise appearance of the DNA-binding RVKNL motif, the WPXXXGAXXFXXL ICP8-binding motif, and the boxIII–boxI palindrome, may have played important roles in establishing major branches of the alphaherpesvirus tree.     

格尔德霉素抗病毒作用的相关基因研究

格尔德霉素(Geldanamycin,GA)作为一种苯醌安莎霉素类抗生素,能与热休克蛋白90特异性结合,具有广谱的抗病毒作用.为了从转录水平上研究GA抗病毒的分子机制,本研究以单纯疱疹病毒Ⅰ型(Herpes simplex vi-rus type 1,HSV-1)为对象,在确定药物有效抗病毒作用的基础上,采用基因芯片技术分析了在HeLa细胞中病毒感染和药物处理对细胞表达谱的影响,并筛选出GA抗病毒作用的可能相关基因.同时用半定量RT-PCR方法对GA诱导上调、HSV-1诱导下调的基因(ACTG1、RAN、SODl)以及GA诱导下调、HSV-1诱导上调的基因(HYALl)进行了验证.研究GA抗病毒作用对细胞表达谱的影响,有利于深入理解药物的抗病毒机制.     

Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

Replication of herpes simplex virus type 1 (HSV-1) involves a step in which a parental capsid docks onto a host nuclear pore complex (NPC). The viral genome then translocates through the nuclear pore into the nucleoplasm, where it is transcribed and replicated to propagate infection. We investigated the roles of viral and cellular proteins in the process of capsid-nucleus attachment. Vero cells were preloaded with antibodies specific for proteins of interest and infected with HSV-1 containing a green fluorescent protein-labeled capsid, and capsids bound to the nuclear surface were quantified by fluorescence microscopy. Results showed that nuclear capsid attachment was attenuated by antibodies specific for the viral tegument protein VP1/2 (UL36 gene) but not by similar antibodies specific for UL37 (a tegument protein), the major capsid protein (VP5), or VP23 (a minor capsid protein). Similar studies with antibodies specific for nucleoporins demonstrated attenuation by antibodies specific for Nup358 but not Nup214. The role of nucleoporins was further investigated with the use of small interfering RNA (siRNA). Capsid attachment to the nucleus was attenuated in cells treated with siRNA specific for either Nup214 or Nup358 but not TPR. The results are interpreted to suggest that VP1/2 is involved in specific attachment to the NPC and/or in migration of capsids to the nuclear surface. Capsids are suggested to attach to the NPC by way of the complex of Nup358 and Nup214, with high-resolution immunofluorescence studies favoring binding to Nup358.     

Neuronal Interferon Signaling Is Required for Protection against Herpes Simplex Virus Replication and Pathogenesis

Interferon (IFN) responses are critical for controlling herpes simplex virus 1 (HSV-1). The importance of neuronal IFN signaling in controlling acute and latent HSV-1 infection remains unclear. Compartmentalized neuron cultures revealed that mature sensory neurons respond to IFNβ at both the axon and cell body through distinct mechanisms, resulting in control of HSV-1. Mice specifically lacking neural IFN signaling succumbed rapidly to HSV-1 corneal infection, demonstrating that IFN responses of the immune system and non-neuronal tissues are insufficient to confer survival following virus challenge. Furthermore, neurovirulence was restored to an HSV strain lacking the IFN-modulating gene, γ34.5, despite its expected attenuation in peripheral tissues. These studies define a crucial role for neuronal IFN signaling for protection against HSV-1 pathogenesis and replication, and they provide a novel framework to enhance our understanding of the interface between host innate immunity and neurotropic pathogens.