Suppression of PACT-Induced Type I Interferon Production by Herpes Simplex Virus 1 Us11 Protein

Herpes simplex virus 1 (HSV-1) Us11 protein is a double-stranded RNA-binding protein that suppresses type I interferon production through the inhibition of the cytoplasmic RNA sensor RIG-I. Whether additional cellular mediators are involved in this suppression remains to be determined. In this study, we report on the requirement of cellular double-stranded RNA-binding protein PACT for Us11-mediated perturbation of type I interferon production. Us11 associates with PACT tightly to prevent it from binding with and activating RIG-I. The Us11-deficient HSV-1 was indistinguishable from the Us11-proficient virus in the suppression of interferon production when PACT was compromised. More importantly, HSV-1-induced activation of interferon production was abrogated in PACT knockout murine embryonic fibroblasts. Our findings suggest a new mechanism for viral evasion of innate immunity through which a viral double-stranded RNA-binding protein interacts with PACT to circumvent type I interferon production. This mechanism might also be used by other PACT-binding viral interferon-antagonizing proteins such as Ebola virus VP35 and influenza A virus NS1.     

敲减单纯疱疹病毒Ⅱ型（HSV-2）UL30蛋白表达可抑制HSV-2复制

按照shRNA（small hairpin RNA）设计要求,选择编码单纯疱疹病毒Ⅱ型DNA多聚酶催化亚单位的U_L30（unique long 30,U_L30）基因序列保守区域,设计、合成并构建表达U_L30序列特异性siRNA（short interfering RNA）的质粒载体pUL30．通过磷酸钙转染法将其转染入HEK（human embryonic kidney）293细胞中,用蛋白印迹法检测对HSV-2 UL30蛋白表达的影响,观察受染细胞病变效应（cytopathic effect,CPE）,终点滴定法测定细胞上清液中病毒感染滴度（50% tissue culture infective dose,TCID₅₀）．结果表明,针对U_L30基因的siRNA能有效抑制UL30蛋白表达,同时显著抑制受染细胞的CPE,降低上清液中病毒感染滴度．提示本研究建立的针对U_L30基因特异性siRNA能有效阻断HSV-2在HEK293细胞内的复制,U_L30基因是一个潜在的抗HSV-2复制的药物靶标．     

Tsg101 Interacts with Herpes Simplex Virus 1 VP1/2 and Is a Substrate of VP1/2 Ubiquitin-Specific Protease Domain Activity

Ubiquitination/deubiquitination of key factors represent crucial steps in the biogenesis of multivesicular body (MVB) and sorting of transmembrane proteins. We and others previously demonstrated that MVB is involved in herpes simplex virus 1 (HSV-1) envelopment and budding. Here, we report that the HSV-1 large tegument protein, VP1/2, interacts with and regulates the ubiquitination of Tsg101, a cellular protein essential in MVB formation, thus identifying the first cellular substrate of a herpesviral deubiquitinating enzyme.     

Regulated Interaction of Tegument Proteins UL16 and UL11 from Herpes Simplex Virus

It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16(1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.     

Herpes Simplex Virus 1 Counteracts Viperin via Its Virion Host Shutoff Protein UL41

The interferon (IFN)-inducible viperin protein restricts a broad range of viruses. However, whether viperin plays a role during herpes simplex virus 1 (HSV-1) infection is poorly understood. In the present study, it was shown for the first time that wild-type (WT) HSV-1 infection couldn''t induce viperin production, and ectopically expressed viperin inhibited the replication of UL41-null HSV-1 but not WT viruses. The underlying molecular mechanism is that UL41 counteracts viperin''s antiviral activity by reducing its mRNA accumulation.     

单纯疱疹病毒1型DNA聚合酶辅助亚基UL42的研究进展

单纯疱疹病毒1型(Herpes simplex virus type 1, HSV-1) UL42作为病毒编码的DNA聚合酶辅助亚基之一，是一种多功能蛋白，其在催化和调节病毒在细胞核内的有效复制发挥了重要的作用。已知UL42能提高DNA聚合酶催化亚基UL30的持续合成能力，激活病毒DNA聚合酶活性；介导DNA聚合酶的入核；与DNA模板链结合，提高病毒复制的保真度，以及含有抑制DNA聚合酶活性的肽段，提示其在病毒复制过程中也可能具有负调控作用。近期亦有报道显示，UL42能够阻断肿瘤坏死因子α(tumor necrosis factor-α， TNF-α)激活的核转录因子(nuclear factor kappa-B，NF-κB)信号通路以及干扰素调控因子3(interferon regulatory factor 3, IRF-3)的功能，提示其在病毒逃逸宿主天然免疫反应中发挥了一定的功能，但具体的作用机制尚不明确。本文对目前国内外HSV-1 UL42的结构特点、主要功能、作用机制及其在抗病毒药物研发中的研究进展进行综述，为后续揭示病毒致病机制和抗病毒药物的研发提供参考。     

Intracellular Trafficking of the UL11 Tegument Protein of Herpes Simplex Virus Type 1

Growing evidence indicates that herpes simplex virus type 1 (HSV-1) acquires its final envelope in the trans-Golgi network (TGN). During the envelopment process, the viral nucleocapsid as well as the envelope and tegument proteins must arrive at this site in order to be incorporated into assembling virions. To gain a better understanding of how these proteins associate with cellular membranes and target to the correct compartment, we have been studying the intracellular trafficking properties of the small tegument protein encoded by the U(L)11 gene of HSV-1. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. When expressed in the absence of other HSV-1 proteins, the UL11 protein localizes to the Golgi apparatus, and previous deletion analyses have revealed that the membrane-trafficking information is contained within the first 49 amino acids. The goal of this study was to map the functional domains required for proper Golgi membrane localization. In addition to N-terminal myristylation, which allows for weak membrane binding, UL11 appears to be palmitylated on one or more of three consecutive N-terminal cysteines. Using membrane-pelleting experiments and confocal microscopy, we show that palmitylation of UL11 is required for both Golgi targeting specificity and strong membrane binding. Furthermore, we found that a conserved acidic cluster within the first half of UL11 is required for the recycling of this tegument protein from the plasma membrane to the Golgi apparatus. Taken together, our results demonstrate that UL11 has highly dynamic membrane-trafficking properties, which suggests that it may play multiple roles on the plasma membrane as well as on the nuclear and TGN membranes.     

Mutational Analysis of the Herpes Simplex Virus Type 1 DNA Packaging Protein UL33

The UL33 protein of herpes simplex virus type 1 (HSV-1) is thought to be a component of the terminase complex that mediates the cleavage and packaging of viral DNA. In this study we describe the generation and characterization of a series of 15 UL33 mutants containing insertions of five amino acids located randomly throughout the 130-residue protein. Of these mutants, seven were unable to complement the growth of the UL33-null virus dlUL33 in transient assays and also failed to support the cleavage and packaging of replicated amplicon DNA into capsids. The insertions in these mutants were clustered between residues 51 and 74 and between 104 and 116, within the most highly conserved regions of the protein. The ability of the mutants to interact with the UL28 component of the terminase was assessed in immunoprecipitation and immunofluorescence assays. All four mutants with insertions between amino acids 51 and 74 were impaired in this interaction, whereas two of the three mutants in the second region (with insertions at positions 111 and 116) were not affected. These data indicate that the ability of UL33 to interact with UL28 is probably necessary, but not sufficient, to support viral growth and DNA packaging.During the packaging of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1), the cleavage of replicated concatemeric viral DNA into single-genome lengths is tightly coupled to its insertion into preassembled spherical procapsids. Upon genome insertion, the internal scaffold protein of the procapsid is lost, and the capsid shell angularizes. Genetic analysis has revealed that successful packaging requires a cis-acting DNA sequence (the a sequence) together with seven proteins, encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes (6, 10). By analogy with double-stranded bacteriophage, the encapsidation of HSV-1 DNA is thought to be mediated by a heteromultimeric terminase enzyme. It is envisaged that the terminase is involved in the recognition of packaging signals present in the concatemers and the association with procapsids via an interaction with the capsid portal protein. Terminase initiates packaging by cleaving at an a sequence present between adjacent genomes within concatemers and subsequently provides energy for genome insertion through the hydrolysis of ATP. Packaging is terminated by a second cleavage event at the next similarly orientated a sequence, resulting in the encapsidation of a unit-length genome.An accumulating body of evidence suggests that the HSV-1 terminase is comprised of the UL15, UL28, and UL33 gene products. Viruses lacking a functional version of any of these three proteins are unable to initiate DNA packaging, and uncleaved concatemers and abortive B-capsids (angularized forms containing scaffold but no DNA) accumulate in the nuclei of infected cells (2, 4, 5, 11, 25, 27, 30, 36, 38). Protein sequence comparisons revealed a distant relationship between UL15 and the large subunit of bacteriophage T4 terminase, gp17, including the presence of Walker A and B box motifs characteristic of ATP binding proteins (13). Subsequent experiments demonstrated that point mutations affecting several of the most highly conserved residues abolished the ability of the resulting mutant viruses to cleave and package viral DNA (26, 39). The UL28 component has been reported to interact with the viral DNA packaging signal (3), a property shared with the homologous protein of human cytomegalovirus (CMV), UL56 (9). Furthermore, both UL15 and UL28 are able to interact with UL6 (33, 37), which form a dodecameric portal complex through which DNA is inserted into the capsid (22, 23, 31). Within the terminase complex, strong interactions have previously been reported between UL15 and UL28 and between UL28 and UL33 (1, 7, 17, 19, 34). Evidence also suggests that UL15 and UL33 may be able to interact directly, albeit more weakly than UL28 and UL33 (7, 15). Temperature-sensitive (ts) lesions in UL33 or UL15 reduced both the interaction of the thermolabile protein with the other members of the terminase complex and viral growth at the nonpermissive temperature (36). Recent evidence suggests that the terminase complex assembles in the cytoplasm and is imported into the nucleus via a mechanism involving a nuclear localization signal within UL15 (35). UL15 is also necessary for the localization of the terminase to nuclear sites of DNA replication and packaging (15). At present, the enzymatic activities necessary for DNA packaging have not been demonstrated for either the complex or individual subunits of the HSV-1 terminase.This study concerns the UL33 protein, which, at 130 residues, is the smallest subunit of the presumptive terminase (7, 27). No specific role in terminase activity has yet been ascribed to UL33, but several possibilities have been proposed including (i) ensuring correct folding or assembly of the complex, (ii) regulating the functions of the other subunits, (iii) performing an essential enzymatic role per se, and (iv) ensuring correct localization of the terminase to sites of DNA packaging (7). However, recent immunofluorescence studies using mutants with defects in the individual terminase subunits suggest that UL33 is unlikely to be involved in this last function (15).In order to further investigate the role of UL33 in the cleavage-packaging process, we utilized transposon-mediated mutagenesis to introduce insertions of five codons throughout the UL33 ORF. We report the generation and characterization of 15 mutants in terms of their ability to support viral growth and DNA packaging and to interact with the terminase component UL28.     

Interaction Domains of the UL16 and UL21 Tegument Proteins of Herpes Simplex Virus

The UL16 protein of herpes simplex virus is capsid associated and was previously identified as a binding partner of the membrane-associated UL11 tegument protein (J. S. Loomis, R. J. Courtney, and J. W. Wills, J. Virol. 77:11417-11424, 2003). In those studies, a less-prominent, ∼65-kDa binding partner of unknown identity was also observed. Mass spectrometry studies have now revealed this species to be UL21, a tegument protein that has been implicated in the transport of capsids in the cytoplasm. The validity of the mass spectrometry results was tested in a variety of coimmunoprecipitation and glutathione S-transferase pull-down experiments. The data revealed that UL21 and UL16 can form a complex in the absence of other viral proteins, even when the assays used proteins purified from Escherichia coli. Moreover, UL11 was able to pull down UL21 only when UL16 was present, suggesting that all three proteins can form a complex. Deletion analyses revealed that the second half of UL21 (residues 268 to 535) is sufficient for the UL16 interaction and packaging into virions; however, attempts to map a subdomain of UL16 were largely unsuccessful, with only the first 40 (of 373) residues being found to be dispensable. Nevertheless, it is clear that UL16 must have two distinct binding sites, because covalent modification of its free cysteines with N-ethylmaleimide blocked binding to UL11 but not UL21. These findings should prove useful for elucidating the molecular machinery used to transmit a signal into a virion when it attaches to cells, a recently discovered mechanism in which UL16 is a central player.Herpes simplex virus (HSV) contains more than 40 different virally encoded proteins that are found in three distinct layers: the capsid containing the viral DNA, the host-derived lipid envelope with embedded glycoproteins, and the tegument, an assortment of proteins located between the nucleocapsid and the envelope (22). While these regions are often discussed as separate structures, there is now clear evidence that the virion as a whole is a machine with interconnected parts that quickly rearrange on the inside in response to glycoprotein-binding events on the outside. Specifically, tegument protein UL16 is triggered to be released from the capsid when HSV attaches to host cells prior to membrane fusion, and the signal responsible for this can be sent in a cell-free manner by binding virions to immobilized heparin (21). It appears that glycoprotein C is involved in transmitting the signal (at least in a cell-free system), but all the other molecular “cogs” that drive this part of the HSV machine are unknown. To identify these components, we have been investigating UL16 and the network of molecular interactions in which it participates.Our interest in UL16 began when we identified it as a binding partner of UL11 (17), a small tegument protein (only 96 amino acids) that is conserved among all herpesviruses. UL11 is peripherally bound to membranes via two fatty acids, myristate and palmitate (16), and trafficks through lipid raft domains (6, 12). It accumulates at the trans-Golgi network (TGN), where virus budding takes place (16, 30), and mutants that lack UL11 are defective for the production of virions, resulting in an increased number of unenveloped capsids in the cytoplasm (5, 9, 19). The UL11-UL16 interaction has since been confirmed by other groups (15, 37), and more recently, we have found that the interaction is direct and requires free cysteines present within UL16 (41). That is, chemical modification of free cysteines in UL16 with N-ethylmaleimide (NEM) blocks the interaction with UL11. On the UL11 side of the interaction, LI and acidic cluster motifs are needed for binding (17, 41).UL16 is a 373-amino-acid protein that is also conserved among herpesviruses and exhibits dynamic capsid-binding properties. Although it is found in both the cytoplasm and the nucleus of the infected cell, it is only stably associated with capsids isolated from the cytoplasm (20, 24, 26). This finding, combined with the ability of UL11 to accumulate at the site of budding, led us to hypothesize that the UL11-UL16 interaction provides a bridging function to assist the capsid in acquiring its envelope (17). However, sometime after budding—as the virus egresses from the cell—the interaction of UL16 with the capsid is destabilized (20). And, as mentioned earlier, binding of the virion to its attachment receptors on the host cell surface (heparan sulfate) further disrupts the association of UL16 with the capsid (21). Free cysteines appear to play a critical role in this outside-in signaling event, because treatment of extracellular virions with NEM prior to cell binding prevents the release of UL16 from the capsid (21).While UL16 was the most abundant protein pulled out of infected cell lysates in our search for UL11 binding partners, a much less prominent, but highly reproducible, ∼65-kDa species was also observed (17). Like UL16, this unknown protein was absent when either the LI or acidic cluster motifs were eliminated from the glutathione S-transferase (GST)-UL11 construct used in the experiment. This suggested that the unknown protein was obtained by either (i) competing with UL16 for binding to the same motifs within UL11 or (ii) binding to UL11 indirectly through an interaction with UL16. Because the LI and acidic cluster motifs of UL11 are recognized by host proteins for trafficking through lipid rafts (6, 16), the first hypothesis seemed likely; however, because UL16 participates in a complex signaling pathway within the virion, it was possible that the unknown protein would be a virus-encoded component. The purpose of the experiments described in this report was to identify this unknown protein and to determine how it fits into the UL16 network of interactions.     

单纯疱疹病毒Ⅰ型新开放读码框UL57的鉴定

单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)潜伏感染期间LATs的活跃转录可能与其启动子与增强子两侧的CTCF结合序列有关。本研究对位于UL56下游与LAT启动子上游之间并与CTCF结合序列重叠存在的一个新开放读码框(本研究中命名为UL57)进行了鉴定。首先利用HSV-1(F)细菌人工染色体(HSV-BAC)系统构建重组病毒HSV-EGFP-UL57,将EGFP序列插入UL57 5’端;然后分别通过Northern Blot和Western Blot检测EGFP标记的UL57的转录和表达;同时构建敲除UL57的重组病毒HSV-ΔUL57,观察UL57对病毒增殖的影响。结果显示,重组病毒HSV-EGFP-UL57感染HEp-2细胞17h后,EGFP探针检测到两条转录产物,其中1.8kb转录产物与预测大小相符;使用放线菌酮(Cycloheximide,CHX)阻断病毒即刻早期蛋白/早期蛋白合成后,UL57转录受到明显抑制。重组病毒HSV-EGFP-UL57感染Vero细胞后,9h可见融合蛋白表达,24h表达明显;融合蛋白分子量与预测大小(58kD)一致。病毒生长曲线显示,重组病毒HSV-EGFP-UL57及HSV-ΔUL57在Vero细胞中的增殖水平与HSV-1(F)基本一致。本研究表明,在HSV-1基因组(GenBank:GU734771.1)UL56下游与LAT启动子上游之间存在一个新开放读码框UL57(116 921bp～117 799bp),UL57可以进行转录,且其转录受病毒即刻早期蛋白/早期蛋白调控;转录产物可以翻译出融合蛋白,但表达水平较低。删除UL57对病毒增殖无明显影响。     

The Unusual Fold of Herpes Simplex Virus 1 UL21, a Multifunctional Tegument Protein

UL21 is a conserved protein in the tegument of alphaherpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis. To provide a roadmap for exploration of the multiple roles of UL21, we determined the crystal structure of its conserved N-terminal domain from herpes simplex virus 1 to 2.0-Å resolution, which revealed a novel sail-like protein fold. Evolutionarily conserved surface patches highlight residues of potential importance for future targeting by mutagenesis.     

Hepatitis A Virus 3C Protease Cleaves NEMO To Impair Induction of Beta Interferon

NEMO (NF-κB essential modulator) is a bridging adaptor indispensable for viral activation of interferon (IFN) antiviral response. Herein, we show that hepatitis A virus (HAV) 3C protease (3C^pro) cleaves NEMO at the Q304 residue, negating its signaling adaptor function and abrogating viral induction of IFN-β synthesis via the retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5) and Toll-like receptor 3 (TLR3) pathways. NEMO cleavage and IFN antagonism, however, were lost upon ablation of the catalytic activity of 3C^pro. These data describe a novel immune evasion mechanism of HAV.     

Herpes Simplex Virus 2 UL45 Is a Type II Membrane Protein

In addition to eleven glycoproteins, the herpes simplex virus type 2 (HSV-2) genome encodes several proteins with potential membrane-spanning segments but no asparagine-linked carbohydrates. One of these is UL45. Fractionation of infected cells showed that HSV-2 UL45 is an integral membrane protein, and analysis of UL45 mutants with potential glycosylation sites showed that it has a type II membrane orientation, the first HSV protein known to have this orientation. Furthermore, it is detectable in infected cells at a time similar to that when glycoproteins gB and gD are detected, consistent with a role in cell-cell fusion, which has previously been found for HSV-1 UL45.     

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein

Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance.     

Overexpression of the Ubiquitin-Specific Protease 7 Resulting from Transfection or Mutations in the ICP0 Binding Site Accelerates Rather than Depresses Herpes Simplex Virus 1 Gene Expression

Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.