Insertion Mutations in Herpes Simplex Virus 1 Glycoprotein H Reduce Cell Surface Expression,Slow the Rate of Cell Fusion,or Abrogate Functions in Cell Fusion and Viral Entry

Of the four required herpes simplex virus (HSV) entry glycoproteins, the precise role of gH-gL in fusion remains the most elusive. The heterodimer gH-gL has been proposed to mediate hemifusion after the interaction of another required glycoprotein, gD, with a receptor. To identify functional domains of HSV-1 gH, we generated 22 randomized linker-insertion mutants. Analyses of 22 gH mutants revealed that gH is relatively tolerant of insertion mutations, as 15 of 22 mutants permitted normal processing and transport of gH-gL to the cell surface. gH mutants that were not expressed well at the cell surface did not function in fusion or viral entry. The screening of gH mutants for function revealed the following: (i) for wild-type gH and some gH mutants, fusion with nectin-1-expressing target cells occurred more rapidly than with herpesvirus entry mediator (HVEM)-expressing target cells; (ii) some gH mutants reduced the rate of cell fusion without abrogating fusion completely, indicating that gH may play a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion; and (iii) only one gH mutant, located within the short cytoplasmic tail, completely abrogated function, indicating that the gH cytoplasmic tail is crucial for cell fusion and viral infectivity.Herpes simplex virus (HSV), an enveloped neurotropic virus, infects target cells via membrane fusion, a process executed by viral fusion proteins capable of inserting into target membranes. Unlike many enveloped viruses that induce fusion through the activity of a single viral fusion protein, HSV requires four glycoproteins, glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL), to execute fusion (6, 40, 42). The focus of this study, gH, is expressed as a heterodimer with gL (gH-gL). HSV gH and gL rely on one another for proper folding, posttranslational processing, and transport to the cell and virion surface (5, 23, 35).A sequential model of entry is the prevailing working hypothesis of HSV entry (1-3, 28, 32, 41). Viral attachment is mediated by the binding of glycoprotein C (gC) or gB to cell surface glycosaminoglycans such as heparan sulfate (38). The subsequent fusion between the virion envelope and host cell membrane is thought to result from a series of concerted events. First, gD binds to one of its host cell receptors. These receptors include herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and heparan sulfate modified by specific 3-O-sulfotransferases (39).It was previously proposed that gD binding a receptor induces a conformational change that allows for interactions between gD, gB, and/or gH-gL (1, 2, 8, 10, 16, 25, 32). It is thought that while gD functions primarily in receptor binding, gB and gH-gL function as the core fusion machinery of HSV.Based on its crystal structure, gB has structural features typical of viral fusion proteins in general and is structurally similar to vesicular stomatitis virus (VSV) glycoprotein G, the fusion protein of VSV (22, 34). In addition to its resemblance to other viral fusogens, gB also binds its own receptor, paired immunoglobulin-like receptor (PILRalpha) (36, 37). Importantly, HSV gB does not successfully execute fusion in the absence of gD or gH-gL (41). Compared to the other required HSV entry glycoproteins, relatively little is known about the specific roles of gH-gL during fusion. The structure of gH-gL is unknown, although in silico analyses and studies of synthetic gH peptides suggested that gH also has fusogenic properties (12, 13, 17-20).gD, a gD receptor, and gH-gL have been shown to be sufficient for inducing hemifusion, the mixing of the proximal leaflets of the viral and host cell bilayers (41). Several lines of research suggest that the subsequent step in fusion is an interaction between gH-gL and gB, with the latter glycoprotein being required for a committed and expanding fusion pore (1-3, 16, 28, 41). However, it is still unclear whether the gB and gH-gL interaction requires that gD first bind a receptor (1, 3), indicating that another viable model of HSV entry may be nonsequential gD-gB-gH-gL complex formation.Several domains important for fusion within HSV gH have been discerned. The only function associated with the N-terminal domain of HSV gH, to date, is gL binding. Residues 377 to 397 within a predicted alpha-helix in the gH ectodomain are required for cell-cell fusion and complementation of a gH-null virus (18). The mutation of a predicted heptad repeat region spanning residues 443 to 471 abrogated cell-cell fusion (17). Insertion mutations within what has been termed the pretransmembrane region of gH have also been shown to abrogate fusion and viral entry (11). The glycine residue at position 812 within the predicted gH transmembrane domain was shown previously to be important for fusion (21). Finally, although the deletion of the final six residues of gH (residues 832 to 838), which are within its short cytoplasmic tail, has no effect on fusion, further deletions were shown to decrease polykaryocyte formation by a syncytial HSV strain (4, 43).We used a transposon-based comprehensive random linker-insertion mutagenesis strategy to generate a library of mutants spanning the entire length of HSV-1 gH, an 838-amino-acid type I membrane protein. A panel of 22 insertion mutants was generated, 15 of which were expressed at near-normal levels on the cell surface. Interestingly, some insertions reduced the rate of cell fusion rather than abrogating cell fusion activity altogether, suggesting that gH may have a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion. Additionally, one insertion mutation that completely abrogated cell fusion and viral infectivity is located within the gH cytoplasmic tail, indicating that the short C-terminal tail of gH is critical for cell fusion and entry mediated by HSV-1.     

Analysis of Herpes Simplex Virus Type 1 DNA Packaging Signal Mutations in the Context of the Viral Genome

The minimal signal required for the cleavage and packaging of replicated concatemeric herpes simplex virus type 1 (HSV-1) DNA corresponds to an approximately 200-bp fragment, Uc-DR1-Ub, spanning the junction of the genomic L and S segments. Uc and Ub occupy positions adjacent to the L and S termini and contain motifs (pac2 and pac1, respectively) that are conserved near the ends of other herpesvirus genomes. We have used homologous Red/ET recombination in Escherichia coli to introduce wild-type and specifically mutated Uc-DR1-Ub fragments into an ectopic site of a cloned HSV-1 genome from which the resident packaging signals had been previously deleted. The resulting constructs were transfected into mammalian cells, and their abilities to replicate and become encapsidated, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus were assessed. In general, the results obtained agree well with previous observations made using amplicons and confirm roles for the pac2 T element in the initiation of DNA packaging and for the GC-rich motifs flanking the pac1 T element in termination. In contrast to a previous report, the sequence of the DR1 element was also crucial for DNA packaging. Following repair of the resident packaging signals in mammalian cells, recombination occurred at high frequency in progeny virus between the repaired sequences and mutated Uc-DR1-Ub inserts. This restored the ability of mutated Uc-DR1-Ub inserts to generate terminal fragments, although these were frequently larger than expected from simple repair of the original lesion.Herpesviruses possess linear double-stranded DNA genomes that are circularized early after infection and upon replication generate concatemeric structures. During progeny particle assembly, the cleavage of concatemers at specific sites, corresponding to the genomic termini, is tightly coupled to the insertion of the viral DNA into a preformed structure referred to as the procapsid (reviewed in references 2, 4, and 11). In the case of herpes simplex virus type 1 (HSV-1), a terminally redundant region of the genome, known as the a sequence (Fig. ​(Fig.1a),1a), contains all the cis-acting sequences required for DNA packaging (24, 27). This region, which is 250 to 500 bp in length depending on the virus strain, is present as a single copy at the S terminus and as one or more tandem copies at the L terminus. In addition, one or more copies are present in inverted orientation at the junction between the L and S segments (30, 31).Open in a separate windowFIG. 1.Structure of the HSV-1 Uc-DR1-Ub element. (a) Structure of the HSV-1 genome showing the positions and relative orientations (horizontal arrows) of copies of the a sequence. (b) Circularization of linear genomes by direct ligation brings together two copies of the a sequence separated by a single DR1 repeat. The site of ligation, and of cleavage of concatemers, is shown by the vertical arrow. (c) Motifs and regions within the 194-bp Uc-DR1-Ub fragment. To facilitate naming of mutants, component regions of Uc, Ub, and DR1 were also referred to as c1 to c4, b1 to b4, and R, respectively, as indicated in parentheses.The structure of the HSV-1 a sequence is depicted in Fig. ​Fig.1b.1b. Each a sequence is flanked by direct repeats (DR1) of 17 to 20 bp, with single copies of DR1 separating tandem a sequences. Genomic termini are generated by a cleavage event toward one end of DR1, and circularization of infecting genomes restores a complete a sequence. The central portion of the a sequence comprises multiple repeats of one or two other short sequences (DR2 and sometimes DR4), while quasi-unique sequences are located between DR1 and either side of the DR2/DR4 repeats. These regions are termed Ub and Uc, and in virion DNA they lie adjacent to the S and L termini, respectively (6, 17, 18).An approximately 200-bp fragment (Uc-DR1-Ub) spanning the junction between tandem a sequences, such as is generated upon fusion of the genomic ends (Fig. ​(Fig.1b),1b), has been shown to contain all the essential cis-acting sequences necessary for DNA packaging (10, 20). Within the Ub and Uc regions are two domains, pac1 and pac2, respectively, which contain several characteristic sequence motifs that are conserved near the ends of other herpesvirus genomes (3, 8, 15). These motifs, as originally defined by Deiss et al. (8), are illustrated in Fig. ​Fig.1c.1c. It is now recognized that the major conserved motif within the pac1 region comprises the T-rich element flanked on each side by short G tracts (from the proximal and distal GC-rich regions). In the case of pac2, the T-rich element is most highly conserved with a consensus CGCGGCG motif also frequently being present (32).Detailed studies, employing primarily HSV-1 and murine cytomegalovirus (MCMV), have highlighted the roles of the major conserved motifs and suggested the following general mechanism by which concatemers are cleaved and packaged (1, 10, 13, 15, 16, 23, 25, 29, 32). Within Uc the most critical sequence is the pac2 T element, which is essential for cleavage to initiate DNA packaging. Cleavage occurs at a fixed distance from the pac2 T element, and the resulting Uc-containing end is inserted into the procapsid. Additional important cis-acting sequences are present further from the cleavage site, possibly including the pac2 consensus motif. Deletion, but not substitution, of the pac2 GC element and unconserved region impaired DNA packaging, suggesting that the relative spacing of the cleavage site, T element, and distal motifs is crucial. Packaging proceeds from pac2 toward the pac1 terminus, and a second cleavage event terminates DNA packaging. This cleavage appears to be directed by, and occurs at a fixed distance from, a single region comprising the pac1 T element and the flanking G tracts. Surprisingly, substitutions within the highly conserved T element are tolerated, but it remains unclear whether this region functions as a spacer element. The UL28 component of the HSV-1 terminase enzyme binds to a specific conformation adopted by the region comprising the T element and G tracts, and this interaction is likely to be crucial for cleavage.The functional analysis of herpesvirus DNA packaging signals has employed two major approaches. In the first, amplicons (i.e., bacterial plasmids containing a viral DNA replication origin and packaging signal) are transfected into mammalian cells and their ability to be replicated and packaged is assessed following the provision of viral helper functions, either by superinfection with virus particles or by cotransfection of virion DNA (7, 20, 24, 27, 29, 35). The second assay introduces an additional copy of the packaging signal under test at an ectopic site within the viral genome and determines whether it functions as a site for the cleavage of concatemeric DNA and the generation of novel terminal fragments of virion DNA (5, 15, 18, 23, 29, 32). Both these approaches, however, suffer from the disadvantage that recombination occurs between the test packaging signal and the wild-type (wt) signal present either in the helper virus or in its normal location within an ectopic-site recombinant (5, 8, 15, 23, 32). Additionally, concatemers generated following replication of amplicons have a significantly different structure from standard herpesviral genomes in that multiple copies of the packaging signal are present, spaced at regular intervals corresponding to the size of the input plasmid. This raises the possibility that the activity of wt or mutated packaging signals in the amplicon assay may not accurately reflect their behavior in a standard genome.To avoid these difficulties and allow analysis of mutated packaging signals in the context of the viral genome, we have used a cloned full-length HSV-1 genome, fHSVΔpac, which is complete with the exception that all copies of the a sequence have been deleted (22). This molecule is propagated as a bacterial artificial chromosome (BAC), and specific sequences can be inserted via homologous recombination either in mammalian cells or in the bacterial host. We previously demonstrated that a single copy of the minimal packaging signal Uc-DR1-Ub introduced into the viral thymidine kinase (TK) locus of fHSVΔpac by recombination in mammalian cells was sufficient to allow the products of replication to be packaged in mammalian cells and to allow the generation of viable progeny (28). Here, we describe the introduction of the packaging signal into fHSVΔpac by Red/ET recombination in Escherichia coli (19, 34), allowing previously described (10) and new Uc-DR1-Ub mutants to be screened for their ability to direct encapsidation, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

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...     

Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.     

Expression of Herpes Simplex Virus 1 MicroRNAs in Cell Culture Models of Quiescent and Latent Infection

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.     

一种可切除包装信号的重组1型单纯疱疹病毒的产生

A new kind of recombinant herpes simplex virus type 1 (HSV 1) was constructed. This recombinant, named HSV1 LaL, contained an unique packaging signal (“a" sequence) flanked by two loxP sites in parallel orientation, named LaL, while the original packaging signals of HSV 1 were deleted. Based on a set of cosmids containing the entire HSV 1 genome except the “a" sequence, the LaL was inserted into HSV 1 UL44 gene on one of the cosmids, cos56, generating cos56/LaL. By co transfecting cos56/LaL with the other cosmids, HSV1 LaL was generated in the cells by recombination. By introducing cos56/LaL or HSV1 LaL respectively into E.coli or BHK cells that expressed Cre recombinase, LaLs on both of them were excised by Cre, which was proved by PCR detection. To study the potential use as helper virus in packaging amplicon vector, HSV1 LaL was compared with a control virus HSV1 lacZ that contained a lacZ gene in the UL44 gene. The titer of amplicon virus generated from HSV1 LaL infected BHK/Cre cells was basically the same as that from HSV1 lacZ infected cells, however,the former contained about 10 fold less helper virus than the later, while HSV1 LaL showed the same replication rate as HSV1 lacZ on standard cells, like BHK 21.     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别.表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白.重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答.     

Mode of Virus Rescue Determines the Acquisition of VHS Mutations in VP22-Negative Herpes Simplex Virus 1

It has been proposed that herpes simplex virus 1 with VP22 deleted requires secondary mutation of VHS for viability. Here we show that a replication-competent Δ22 virus constructed by homologous recombination maintains a wild-type (Wt) VHS gene and has no other gross mutations. By contrast, Δ22 viruses recovered from a bacterial artificial chromosome contain multiple amino acid changes within a conserved region of VHS. Hence, the mode of virus rescue influences the acquisition of secondary mutations.     

Herpes Simplex Virus Type 1 Infection of Isogenic Epstein-Barr Virus Genome-Negative and -Positive Burkitt''s Lymphoma-Derived Cell Lines

The Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma-derived cell lines BJAB and Ramos and their in vitro EBV-converted sublines BJAB-B1, BJAB-A5, BJAB-B95-8, and AW-Ramos were infected with high multiplicities of herpes simplex virus type 1 (HSV-1; 10 to 70 PFU/cell). Cultures were monitored for cell growth and HSV-1 DNA synthesis. EBV-converted BJAB cultures were more permissive for HSV-1 infection than BJAB cultures. Significant cell killing and HSV-1 DNA synthesis were observed during the first 48 h of infection in the EBV-converted BJAB cultures but not in the BJAB cultures. The EBV-converted BJAB-B1 cell line contains an appreciable fraction of EBV-negative cells. Therefore, it was cloned. EBV-positive and -negative cells were identified by using EBV-determined nuclear antigen anti-complement immunofluorescence. Two types of subclones were identified: (i) those which contained both EBV-determined nuclear antigen-positive and -negative cells and (ii) those which contained only EBV-determined nuclear antigen-negative cells. When levels of HSV-1 DNA synthesis were measured in these subclones, it was found that the former were more permissive for HSV-1 infection than the latter. Thus, the presence of the EBV genome in BJAB cells correlates with increased permissiveness of these cells for HSV-1 during the first 48 h of infection. Nonetheless, persistent HSV-1 infections were established in both BJAB and EBV-converted BJAB-B1 cultures. No differences in extent of permissiveness for HSV-1 infection were found for Ramos and EBV-converted AW-Ramos cells.     

The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.     

Isolation and Characterization of the Herpes Simplex Virus 1 Terminase Complex

During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.     

PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function ofeIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

Genome Sequence of Herpes Simplex Virus 1 Strain McKrae

The herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent compared to other wild-type strains of HSV-1. To help us better understand the genetic determinants that lead to differences in the pathogenicity of McKrae and other HSV-1 strains, we sequenced its genome. Comparing the sequence of McKrae's genome to that of strain 17 revealed that the genomes differ by at least 752 single nucleotide polymorphisms (SNPs) and 86 insertion/deletion events (indels). Although the majority of these polymorphisms reside in noncoding regions, 241 SNPs and 10 indels alter the protein-coding sequences of 58 open reading frames. Some of these variations are expected to contribute to the pathogenic phenotype of McKrae.