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.     

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.     

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.     

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.     

Herpes Simplex Virus Type 1 Tegument Protein VP22 Induces the Stabilization and Hyperacetylation of Microtubules

The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4°C, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.

The eukaryotic cytoskeleton, which comprises actin microfilaments, intermediate filaments (IFs), and microtubules (MTs), performs a broad range of complex activities within the cell. These include various aspects of cell motility (2, 3), the determination of cell shape and internal architecture (17, 32), and vesicle trafficking and chromosome movement during mitosis (18, 25, 29). Furthermore, the individual components of the cytoskeleton are interlinked to form a dynamic network accessing every area of the cytoplasm (41) and the plasma membrane (10, 39), providing a framework which coordinates multiple cellular processes. The involvement in so many cellular activities is likely to make the cytoskeleton a primary target for exploitation during virus infection of host cells. Surprisingly, however, there is relatively little detailed information on virus interactions with the host cytoskeleton, and it is only recently that data suggesting that viruses may utilize the positioning and dynamics of the cytoskeletal network to their own advantage have begun to emerge.The majority of virus-induced cytoskeletal alterations documented to date involve the overall disruption of one or more elements of the cytoskeleton. For example, retroviruses and poliovirus encode proteases which induce the cleavage of cytoskeleton-associated proteins, thereby broadly increasing the dynamics of the cytoskeleton, resulting in disruption of the cell structure as infection progresses, and the appearance of well-characterized cytopathic effects (20, 43). A more specific disruption of the cytoskeleton occurs during infection by the rhabdovirus vesicular stomatitis virus, where the direct interaction of the virus matrix protein with tubulin results in the inhibition of MT assembly (33). Human immunodeficiency virus and papillomaviruses, on the other hand, encode activities which induce the collapse of the IF network, a property which may promote virus release from the cell (13, 23).By contrast, examples of virus activities which induce cytoskeletal polymerization and/or stabilization are much rarer. One example is the baculovirus Autographa californica nuclear polyhedrosis virus, which has been shown to induce the appearance of thick actin cables between the plasma membrane and the nucleus at early times after infection (8) and to induce actin filaments in the nucleus at late times (7). These features have been proposed to be involved in virus transport from the cell surface to the nucleus and nucleocapsid morphogenesis, respectively. However, the best-characterized viral exploitation of the host cell cytoskeleton is that of vaccinia virus, which has been shown to induce actin polymerization directly behind its virus particle as a means of propelling the virus through the cell (11, 12). The virus protein(s) responsible for this activity has not yet been identified, but it has been shown that disruption of the actin cytoskeleton in infected cells inhibits virus release, indicating that actin is essential to the virus replicative cycle (35).The herpes simplex virus type 1 (HSV-1) structural protein VP22, a component of the viral tegument, has an as yet undefined role in virus replication. However, we have recently shown that VP22 has the unusual property of intercellular transport when it is expressed during both infection and transient transfection (14). Moreover, we demonstrated that such VP22 transport occurs via a mechanism potentially involving actin microfilaments, suggesting that VP22 exhibits a cytoskeletal interaction. In this report, we demonstrate that VP22 interacts with another component of the cellular cytoskeleton, the MT network. We show that VP22 colocalizes with MTs in both transfected and infected cells and induces the appearance of thick MT bundles. Furthermore, we show that these VP22-induced MT bundles are highly stabilized in comparison to normal MTs and are resistant to both drug and cold treatment. As a consequence of VP22-induced stabilization, MTs are extensively modified by acetylation, a property also demonstrated in infected cells. Taken together, these results suggest that VP22 exhibits the properties of a classical cellular MT-associated protein (MAP) with powerful MT-stabilizing properties and represents the first demonstration of a MAP encoded by an animal virus.     

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.     

Phosphorylation of Structural Components Promotes Dissociation of the Herpes Simplex Virus Type 1 Tegument

The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.     

A Network of Protein Interactions around the Herpes Simplex Virus Tegument Protein VP22

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22''s major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.     

Identification of Phosphorylation Sites within the Herpes Simplex Virus Tegument Protein VP22

The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.     

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction wit...     

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.     

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.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

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。     

The Herpes Simplex Virus Type 1 UL17 Gene Encodes Virion Tegument Proteins That Are Required for Cleavage and Packaging of Viral DNA

Previous studies have suggested that the U_L17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U_L17 open reading frame [HSV-1(ΔU_L17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the U_L17 open reading frame [HSV-1(U_L17-stop)] were plaque purified on engineered cell lines containing the U_L17 gene. A virus derived from HSV-1(U_L17-stop) but containing a restored U_L17 gene was also constructed and was designated HSV-1(U_L17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔU_L17) nor HSV-1(U_L17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔU_L17) or HSV-1(U_L17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔU_L17) or HSV-1(U_L17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔU_L17) compared to wild-type virus show no detectable differences. These data indicate that the U_L17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U_L17 gene product, an anti-U_L17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent M_r 77,000 and weakly with a protein of apparent M_r 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U_L17 protein. We therefore conclude that the U_L17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.