Synthesis, solution structure analysis and antibody binding of cyclic epitope peptides from glycoprotein D of Herpes simplex virus type I

Two cyclic peptides with a thioether bond have been synthesised corresponding to the 9-22 (9LKMADPNRFRGKDL(22)) sequence of glycoprotein D (gD-1) of Herpes simplex virus. The role of the secondary structure in protein-specific monoclonal antibody recognition was investigated. The sequence selected for this study comprises a strongly antigenic site adopting a beta-turn at residues 14Pro-(15)Asn. Thioether bond was formed between the free thiol group of cysteine or homocysteine inserted in position 11 and the chloroacetylated side chain of lysine in position 18. We report here the preparation of cyclic peptides containing Cys or Hcy in position 11, differing only in one methylene group. The linear precursor peptides were synthesised by Boc/Bzl strategy on MBHA resin, and the cyclisation was carried out in alkaline solution. The secondary structure of the peptides was studied by CD, FT-IR and NMR spectroscopy. The CD and FT-IR data have revealed fundamental changes in the solution conformation of the two compounds. The CH(2) group difference significantly resulted in the altered turn structure at the 12Ala and 13Asp as identified by NMR spectroscopy. The antibody binding properties of the cyclopeptides studied by gD-specific monoclonal antibody (A16) in direct and competition enzyme-linked immunosorbent assay (ELISA) were also not the same. We found that peptide LK[HcyADPNRFK]GKDL exhibited higher affinity to Mab A16 than peptide LK[CADPNRFK]GKDL, however, their reactivity was significantly lower compared to the linear ones. Our results clearly show the importance of secondary structure in an antibody binding and demonstrate that even a slight modification of the primary structure dramatically could influence the immune recognition of the synthetic antigens.     

Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein

Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced widely enough (at least 32 A) that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.     

Effect of conjugation with polypeptide carrier on the enzymatic degradation of Herpes simplex virus glycoprotein D derived epitope peptide

Two conjugates with epitope peptide (278)LLEDPVGTVA (287) derived from glycoprotein D (gD-1) of Herpes simplex virus (HSV) were synthesized for analysis of the effect of conjugation on protection against enzymatic degradation. In this design, the turn-forming epitope core (281)DPVG (284) was positioned in the central part of the peptide and elongated by three amino acids from the native sequence at both termini. Conjugation was achieved by the introduction of amide bond or thioether linkage between the C-terminal of the HSV peptide and the side chain of four lysine residues of the oligotuftsin derivative used as carrier molecule. We compared the proteolytic stability of the conjugates in diluted human sera as well as in rat liver lysosomal preparation. The data obtained in lysosomal preparation at two pH values (pH 3.5 and 5.0) show that the type of covalent bond between the carrier and the epitope peptide had no significant effect, as compared to the stability of the free, unconjugated peptide. Based on the identification of degradation fragments by mass spectrometry we found marked differences in the lengths and amounts of oligopeptides obtained. In contrast, in 10% and 50% human serum the conjugation provided full protection against enzymatic hydrolysis over 96 h, while the free peptide was decomposed quickly.     

Herpes simplex virus type 1 glycoprotein K and the UL20 protein are interdependent for intracellular trafficking and trans-Golgi network localization

Final envelopment of the cytoplasmic herpes simplex virus type 1 (HSV-1) nucleocapsid is thought to occur by budding into trans-Golgi network (TGN)-derived membranes. The highly membrane-associated proteins UL20p and glycoprotein K (gK) are required for cytoplasmic envelopment at the TGN and virion transport from the TGN to extracellular spaces. Furthermore, the UL20 protein is required for intracellular transport and cell surface expression of gK. Independently expressed gK or UL20p via transient expression in Vero cells failed to be transported from the endoplasmic reticulum (ER). Similarly, infection of Vero cells with either gK-null or UL20-null viruses resulted in ER entrapment of UL20p or gK, respectively. In HSV-1 wild-type virus infections and to a lesser extent in transient gK and UL20p coexpression experiments, both gK and UL20p localized to the Golgi apparatus. In wild-type, but not UL20-null, viral infections, gK was readily detected on cell surfaces. In contrast, transiently coexpressed gK and UL20p predominantly localized to the TGN and were not readily detected on cell surfaces. However, TGN-localized gK and UL20p originated from endocytosed gK and UL20p expressed at cell surfaces. Retention of UL20p to the ER through the addition of an ER retention motif forced total ER retention of gK, indicating that transport of gK is absolutely dependent on UL20p transport. In all experiments, gK and UL20p colocalized at intracellular sites, including the ER, Golgi, and TGN. These results are consistent with the hypothesis that gK and UL20p directly interact and that this interaction facilitates their TGN localization, an important prerequisite for cytoplasmic virion envelopment and egress.     

Herpes simplex virus type 1-induced modifications in the distribution of nucleolar B-36 protein

Nucleolar B-36 protein was localized ultrastructurally by immunocytochemistry with monoclonal antibody P2G3 and colloidal gold label in rabbit fibroblast cells before and during infection with herpes simplex virus (HSV) type 1. In non-infected cells, labeling was sparse and restricted to the fibrillar component of the nucleoli. During the infectious cycle, B-36 protein appeared to be somewhat more abundant within the morphologically altered fibrillar component of the nucleoli. In addition, the protein was also detected in some but not all virus-induced intranuclear dense bodies. These observations suggest the presence of functionally distinct dense bodies. The association of B-36 protein with both structures was not disrupted by a hypotonic shock and detergent treatment, which suggest that these sites do not represent areas of passive intranuclear diffusion. Inhibition of protein synthesis late in infection, viral DNA replication or RNA synthesis did not alter the distribution of B-36 protein. We suggest that this protein may play a role in the increased compaction of the ribonucleoprotein fibrils induced by HSV infection, perhaps in association with some of the virus-encoded proteins which also have been detected in the nucleoli.     

Low-pH-dependent changes in the conformation and oligomeric state of the prefusion form of herpes simplex virus glycoprotein B are separable from fusion activity

The cellular requirements for activation of herpesvirus fusion and entry remain poorly understood. Low pH triggers change in the antigenic reactivity of the prefusion form of the herpes simplex virus (HSV) fusion protein gB in virions, both in vitro and during viral entry via endocytosis (S. Dollery et al., J. Virol. 84:3759-3766, 2010). However, the mechanism and magnitude of gB conformational change are not clear. Here we show that the conformation and oligomeric state of gB with mutations in the bipartite fusion loops were similarly altered despite the fusion-inactivating mutations. Together with previous studies, this suggests that fusion loop mutants undergo conformational changes but are defective for fusion because they fail to make productive contact with the outer leaflet of the host target membrane. A direct, reversible effect of low pH on the structure of gB was detected by fluorescence spectroscopy. A soluble form of gB containing cytoplasmic tail sequences (s-gB) was triggered by mildly acidic pH to undergo changes in tryptophan fluorescence emission, hydrophobicity, antigenic conformation, and oligomeric structure and thus resembled the prefusion form of gB in the virion. In contrast, soluble gB730, for which the postfusion crystal structure is known, was only marginally affected by pH using these measures. The results underscore the importance of using a prefusion form of gB to assess the activation and extent of conformation change. Further, acidic pH had little to no effect on the conformation or hydrophobicity of gD or on gD's ability to bind nectin-1 or HVEM receptors. Our results support a model in which endosomal low pH serves as a cellular trigger of fusion by activating conformational changes in the fusion protein gB.     

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.     

Characterization of the herpes simplex virus type 1 glycoprotein D mRNA and expression of this protein in Xenopus oocytes

We have identified and characterized a 3.0 kilobase (kb) mRNA containing coding sequences of the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene. The synthesis of this 3.0 kb mRNA was unaffected by the presence of cytosine arabinoside, but was made in greatly reduced amounts in cells infected with HSV-1 in the presence of cycloheximide: it was, therefore, classified as an early mRNA. By nuclease protection experiments, it was found that the 3.0 kb mRNA is unspliced and, further, that it is 3' co-terminal with a smaller 1.6 kb early mRNA which is transcribed from a DNA sequence 3' to the gD coding sequence. We describe the use of the Xenopus laevis oocyte system to produce HSV-1 gD in vitro. Oocytes injected with mRNA isolated from HSV-1-infected Vero cells synthesized gD, which was identified by immunoprecipitation. Injection of a plasmid clone containing the HSV-1 BamHI J fragment (0.89 to 0.93 map units) into the nuclei of Xenopus oocytes also resulted in synthesis of gD.     

Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.     

Herpes simplex virus capsid structure: DNA packaging protein UL25 is located on the external surface of the capsid near the vertices

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.     

The crystal structure of the herpes simplex virus 1 ssDNA-binding protein suggests the structural basis for flexible, cooperative single-stranded DNA binding

All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific, flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 A resolution. The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain (residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees with the beaded morphology observed in electron micrographs.     

Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B

We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCdelta, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCalpha was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCalpha remained in the cytoplasm. PKCzeta-specific immunofluorescence was not significantly relocated to the nuclear rim. The UL34 and UL31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 US3 protein product, a kinase which regulates the phosphorylation state and localization of UL34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with US3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring UL31 and UL34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.     

Herpes simplex virus type 1 single-strand DNA binding protein ICP8 enhances the nuclease activity of the UL12 alkaline nuclease by increasing its processivity

UL12 is a 5'- to 3'-exonuclease encoded by herpes simplex virus type 1 (HSV-1) which degrades single- and double-stranded DNA. UL12 and the single-strand DNA binding protein ICP8 mediate a strand exchange reaction. We found that ICP8 inhibited UL12 digestion of single-stranded DNA but stimulated digestion of double-stranded DNA threefold. The stimulatory effect of ICP8 was independent of a strand exchange reaction; furthermore, the effect was specific to ICP8, as it could not be reproduced by Escherichia coli single-stranded DNA binding protein. The effect of ICP8 on the rate of UL12 double-stranded DNA digestion is attributable to an increase in processivity in the presence of ICP8.     

Crystal structure of a recombinant form of the maltodextrin-binding protein carrying an inserted sequence of a B-cell epitope from the preS2 region of hepatitis B virus

We report the crystal structure of MalE-B133, a recombinant form of the maltodextrin-binding protein (MBP) of Escherichia coli carrying an inserted amino-acid sequence of a B-cell epitope from the preS2 region of the hepatitis B virus (HBV). The structure was determined by molecular replacement methods and refined to 2.7 Å resolution. MalE-B133 is an insertion/deletion mutant of MBP in which residues from positions 134 to 142, an external α helix in the wild-type structure, are replaced by a foreign peptide segment of 19 amino acids. The inserted residues correspond to the preS2 sequence from positions 132 to 145 and five flanking residues that arise from the creation of restriction sites. The conformation of the recombinant protein, excluding the inserted segment, closely resembles that of wild-type MBP in the closed maltose-bound form. MalE-B133 was shown by previous studies to display certain immunogenic and antigenic properties of the hepatitis B surface antigen (HBsAg), which contains the preS2 region. The crystal structure reveals the conformation of the first nine epitope residues (preS2 positions 132 to 140) exposed on the surface of the molecule. The remaining five epitope residues (preS2 positions 141 to 145) are not visible in electron density maps. The path of the polypeptide chain in the visible portion of the insert differs from that of the deleted segment in the structure of wild-type MBP, displaying a helical conformation at positions 134 to 140 (preS2 sequence numbering). A tripeptide (Asp-Pro-Arg) at the N terminus of the helix forms a stable structural motif that may be implicated in the cross-reactivity of anti-HBsAg antibodies with the hybrid protein. Proteins 27:1–8 © 1997 Wiley-Liss, Inc.     

Herpes simplex virus 1 ICP22 regulates the accumulation of a shorter mRNA and of a truncated US3 protein kinase that exhibits altered functions

The U(S)3 open reading frame of herpes simplex virus 1 (HSV-1) was reported to encode two mRNAs each directing the synthesis of the same protein. We report that the U(S)3 gene encodes two proteins. The predominant U(S)3 protein is made in wild-type HSV-1-infected cells. The truncated mRNA and a truncated protein designated U(S)3.5 and initiating from methionine 77 were preeminent in cells infected with a mutant lacking the gene encoding ICP22. Both the wild-type and truncated proteins also accumulated in cells transduced with a baculovirus carrying the entire U(S)3 open reading frame. The U(S)3.5 protein accumulating in cells infected with the mutant lacking the gene encoding ICP22 mediated the phosphorylation of histone deacetylase 1, a function of U(S)3 protein, but failed to block apoptosis of the infected cells. The U(S)3.5 and U(S)3 proteins differ with respect to the range of functions they exhibit.