Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N,a Herpesvirus Serine Protease

Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.     

Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.The herpesvirus virion is composed of an icosahedral nucleocapsid containing the viral genome, an envelope of cellular origin with inserted viral (glyco)proteins, and a tegument which links nucleocapsid and envelope comparable to the matrix of RNA viruses. The herpesvirus tegument contains a multitude of viral and cellular proteins (reviewed in references 45 and 46). Tegument proteins execute various regulatory and structural functions, including activation of viral gene expression (2), modulation of the host cell for virus replication (26, 51, 55), and mediation of posttranslational modification of proteins (10, 27, 50). Numerous interactions have been identified among tegument proteins, between tegument and capsid proteins, and between tegument and envelope proteins (7, 14, 16, 18, 33, 36, 42, 53, 58-61).The largest tegument proteins found in the herpesviruses are homologs of pUL36 of herpes simplex virus type 1 (HSV-1). Pseudorabies virus (PrV) pUL36 consists of 3,084 amino acids (aa) with a molecular mass of 324 kDa (33). PrV and HSV-1 pUL36 are essential for viral replication (13, 15). In their absence, nonenveloped nucleocapsids accumulate in the cytoplasm. Whereas in several studies nuclear stages like cleavage and packaging of the viral DNA as well as nuclear egress were not found affected (13, 15), another study indicated an effect of pUL36 deletion on PrV nuclear egress (41).pUL36 homologs complex with another tegument protein, pUL37, as has been shown for HSV-1 (59), PrV (15, 33), and human cytomegalovirus (3, 23), and the interacting region on pUL36 has been delineated for PrV (33) and identified at the amino acid level for HSV-1 (47). Deletion of the pUL37 interaction domain from PrV pUL36 impedes virion formation in the cytosol but does not block it completely, yielding a phenotype similar to that of a pUL37 deletion mutant (31). This indicates an important but nonessential role for pUL37 and the pUL37 interaction domain in pUL36 in virion formation (15). In contrast, absence of pUL37 completely blocks virion formation in HSV-1 (11, 38).pUL36 is stably attached to the nucleocapsid (39, 43, 56), remains associated with incoming particles during transport along microtubules to the nuclear pore (21, 40, 52), and is required for intracellular nucleocapsid transport during egress (41). In contrast, absence of pUL37 delays nuclear translocation of incoming PrV nucleocapsids but does not abolish it (35). HSV-1 pUL36 is involved not only in transport but also in docking of nucleocapsids to the nuclear pore (9), and proteolytic cleavage of pUL36 appears to be necessary for release of HSV-1 DNA into the nucleus (24).Immunoelectron microscopical studies of PrV-infected cells showed that pUL36 is added to nucleocapsids prior to the addition of pUL37 (33). Since neither pUL36 nor pUL37 was detected on primary enveloped PrV virions, it was concluded that acquisition of tegument takes place in the cytoplasm (20). However, conflicting data exist whether pUL36 is present in the nucleus, and whether it is already added onto the capsids in this cellular compartment. Indirect immunofluorescence, immunoelectron microscopy and mass spectrometry of intranuclear capsids yielded discrepant results. By immunofluorescence HSV-1 pUL36 was detected both in the cytoplasm and in the nucleus (1, 42, 48). However, whereas one study detected the protein on nuclear C-capsids by Western blotting (6), it was not found by cryo-electron microscopy and mass spectrometry (57). In contrast, the C terminus of PrV pUL36 was suggested to direct pUL36 to capsid assemblons in the nucleus (37) by binding to capsid-associated pUL25 (8), although pUL36 could not be detected in the nucleus during PrV infection (33). These differing results in HSV-1 and between HSV-1 and PrV might be due to the fact that pUL36 could be processed during the replication cycle and that the resulting subdomains may exhibit selective localization patterns (24, 28).Amino acid sequence analyses of HSV-1 and PrV pUL36 revealed several putative nuclear localization signals (NLS) (1, 4, 5, 49). HSV-1 pUL36 contains four of these NLS motifs (49). Functionality in nuclear localization of a reporter protein was shown for the NLS motif at aa 425 (1). This motif is highly conserved in herpesvirus pUL36 homologs pointing to an important function (1). Besides this conserved NLS (designated in this report as NLS1), two other NLS motifs are predicted in PrV pUL36. One is located adjacent to NLS1 (aa 288 to 296) at aa 315 to 321 (NLS2), and a third putative NLS motif is present in the C-terminal half of the protein (aa 1679 to 1682; NLS3) (4). Whereas this may be indicative for a role for pUL36 inside the nucleus, NLS motifs might also be involved in transport to the nucleus along microtubules (54) and docking at the nuclear pore complex (49).The discrepancy in pUL36 localization and the putative presence of pUL36 cleavage products with specialized functions and localization prompted us to generate monospecific antisera covering the major part of PrV pUL36 and to study localization of PrV pUL36 by immunofluorescence during viral replication and after transient transfection and by immunoelectron microscopy of infected cells.     

Random Transposon-Mediated Mutagenesis of the Essential Large Tegument Protein pUL36 of Pseudorabies Virus

Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.The herpesvirus particle is composed of four structural elements. The DNA genome-containing core is enclosed in an icosahedral capsid, which, in turn, is embedded in a proteinaceous layer termed the tegument and enveloped by a cell-derived membrane containing viral glycoproteins (35). The tegument of the Alphaherpesvirinae contains more than 15 different viral and several cellular proteins and can be structurally and functionally separated into at least two layers: a capsid-proximal “inner” part and an envelope-associated “outer” part (reviewed in references 34 and 35). The largest tegument proteins in all herpesviruses analyzed so far are homologs of herpes simplex virus type 1 (HSV-1) pUL36, which are essential for viral replication. pUL36, its interaction partner, pUL37, and the pUS3 kinase are part of the inner tegument and remain associated with nucleocapsids during their transport along microtubules to the nuclear pore (2, 3, 19, 31). In contrast, other tegument proteins like pUL46, pUL47, and pUL49 rapidly diffuse in the cytoplasm after fusion of the virion envelope with the plasma membrane. Proteolytic cleavage of HSV-1 pUL36 after docking of the nucleocapsid to the nuclear pore appears to be required for release of viral DNA into the nucleus (22). Besides these roles early in infection, pUL36 also functions during later stages of replication in virion maturation. After assembly in the nucleus, nucleocapsids are translocated to the cytoplasm by budding at the inner nuclear membrane and fusion with the outer nuclear membrane (34). Although functional nuclear localization motifs have been described for pseudorabies virus (PrV) and HSV-1 pUL36 (1, 37), in PrV-infected cells, pUL36 was never detected in the nucleus but was added to nascent virions early after nuclear egress (18, 27, 31, 37). It has been suggested that pUL36 interacts either directly (9, 32, 42, 44) or indirectly via capsid-associated pUL25 (10) with the capsid shell starting the tegumentation process in the cytosol.In PrV, pUL36 is the only tegument protein which has been shown to be truly essential. It consists of 3,084 amino acids (aa), resulting in a molecular mass of more than 300 kDa (27). Deletion of pUL36 in HSV-1 and PrV abolished viral replication. Ultrastructurally, similar phenotypes with nonenveloped nucleocapsids present in the cytoplasm and the lack of extracellular particles indicated a defect in virion maturation in the cytoplasm (13, 16). Several functional domains have been identified in pUL36. The interaction domain of pUL36 with pUL37 (5, 16, 20, 27, 36, 42) could be located in the N-terminal part of PrV and HSV-1 pUL36 (16, 36) (Fig. ​(Fig.1).1). Deletion of the pUL37 binding site in PrV pUL36 (PrV-UL36BSF) resulted in a similar phenotype to deletion of pUL37 with an impairment of secondary envelopment in the cytoplasm (16, 26). Unlike in PrV, pUL37 is essential for replication in HSV-1 (14, 30).Open in a separate windowFIG. 1.Schematic overview of PrV pUL36 and corresponding insertion mutants. (A) Diagram of the PrV genome with the unique long (U_L) and unique short (U_S) regions as well as repeat regions (internal repeat, IR; terminal repeat, TR). The positions of BamHI restriction sites are indicated, and restriction fragments are numbered according to their size. (B) Schematic diagram of the UL36 open reading frame with conserved regions. Pfam analysis (4; http://www.sanger.ac.uk/Software/Pfam/) delineated two highly conserved PfamA domains within pUL36 homologs of herpesviruses of all three herpesvirus subfamilies [box I, Herpes_teg_N PrV (p)UL36, aa 11 to 178] and of alphaherpesviruses [box II, Herpes_UL36 PrV (p)UL36, aa 1000 to 1251] as well as PfamB domains (hatched rectangles) (6) (C) Known essential and nonessential regions in PrV pUL36. Nonessential regions are shown in gray, with the positions of the amino acids deleted in the corresponding constructs (6, 8). Deletions tested by Lee et al. (28) are shown below, marked by arrows. The essential C terminus is shown in black. Besides the N-terminal deletion Δ6-225, none of the truncated proteins was functional. (D) Predicted or identified motifs in pUL36: USP (Cys26), active-site cysteine of the deubiquitinating activity (24); pUL37 interaction domain (16, 27); NLS, nuclear localization signal (37); leucine zipper (27); and late domain motifs PPKY and PSAP (6). (E) Locations of linker insertions in pUL36 are indicated by arrows and the position of the amino acid immediately preceding the insertion. Insertions shown by arrows pointing upwards yielded functional proteins, while arrows pointing downwards indicate nonfunctional mutants. Insertions resulting in proteins which were impaired but not fully deficient in complementation are underlined. For orientation, the BamHI site separating BamHI fragments 1 and 2 is indicated.A second functional domain in the N terminus of pUL36 comprises a ubiquitin-specific cysteine protease (USP) activity which could be identified in all three herpesvirus subfamilies (24, 40, 41). Interestingly, the USP activity is not essential for virus replication in cell culture (7, 21, 25, 43). However, it is relevant for oncogenicity of Marek′s disease virus (MDV) (21) and for virion maturation and neuroinvasion of PrV (7, 8, 29).Several other regions in PrV pUL36 were deleted without abolishing virus replication (6, 8, 28). While deletion of nearly 1/3 of the protein in the C-terminal part (aa 2087 to 2981) had only a slight effect, deletion of a region containing two leucine zipper motifs impaired virus replication and spread more strongly (8). The highly conserved C-terminal 62 amino acids, except for the extreme C-terminal 6 amino acids, are essential for virus replication (6, 28). Due to the size of the protein, a more detailed mutagenesis analysis has, however, not yet been undertaken.Therefore, the aim of our study was to construct random insertion mutants of PrV pUL36 using transposon-mediated insertion mutagenesis resulting in a 5-amino-acid linker insertion. Mutant proteins were analyzed functionally in transient transfection assays for complementation, and stable recombinants were isolated and further characterized.     

Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn⁺²-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn⁺², the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn⁺²-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease.     

Glycoproteins M and N of Pseudorabies Virus Form a Disulfide-Linked Complex

Genes homologous to the herpes simplex virus UL49.5 open reading frame are conserved throughout the Herpesviridae. In the alphaherpesvirus pseudorabies virus (PrV), the UL49.5 product is an O-glycosylated structural protein of the viral envelope, glycoprotein N (gN) (A. Jöns, H. Granzow, R. Kuchling, and T. C. Mettenleiter, J. Virol. 70:1237–1241, 1996). For functional characterization of gN, a gN-negative PrV mutant, PrV-gNβ, and the corresponding rescuant, PrV-gNβR, were constructed, gN-negative PrV was able to productively replicate on noncomplementing cells, and one-step growth in cell culture was only slightly reduced compared to that of wild-type PrV. However, penetration was significantly delayed. In indirect immunofluorescence assays with rabbit serum directed against baculovirus-expressed gN, specific staining of wild-type PrV-infected cells occurred only after permeabilization of cells, whereas live cells failed to react with the antiserum. This indicates the lack of surface accessibility of gN in the plasma membrane of a PrV-infected cell. Western blot analyses and radioimmunoprecipitation experiments under reducing and nonreducing conditions led to the discovery of a heteromeric complex composed of gM and gN. The complex was stable in the absence of 2-mercaptoethanol but dissociated after the addition of the reducing agent, indicating that the partners are linked by disulfide bonds. Finally, gN was absent from gM-negative PrV virions, whereas gM was readily detected in virions in the absence of gN. Thus, gM appears to be required for virion localization of gN.     

一种新的丙型肝炎病毒单链丝氨酸蛋白酶基因的构建、表达与鉴定

根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .     

Identification and Characterization of Fusolisin,the Fusobacterium nucleatum Autotransporter Serine Protease

Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61–65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55–101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96–113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55–65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.     

Channel Catfish Virus: a New Herpesvirus of Ictalurid Fish

Channel catfish virus was studied in ictalurid fish cell culture, the only system of fish, amphibian, avian, and mammalian cells found to be susceptible. Channel catfish virus infection resulted in intranuclear inclusions and extensive syncytium formation. Replication occurred from 10 to 33 C, but not higher. Best growth was from 25 to 33 C, and the amount of virus released nearly equalled the amount which remained cell-associated. The virus was labile to lipid solvents, and indirect determinations with labeled precursors and a metabolic inhibitor showed evidence of deoxyribonucleic acid. Electron microscopy showed progeny virus, about 100 nm in diameter, in various stages of development in cell nuclei by 4 hr. Present also were nuclear masses of exceptionally electron-dense lamellar material, with a unit dimension of 10 to 15 nm. Virus was enveloped at the nuclear membrane and in cytoplasmic vacuoles, resulting in virions having a diameter of 175 to 200 nm. Negative staining demonstrated icosehedral symmetry and 162 capsomeres. Our data indicate that channel catfish virus is a herpesvirus.     

Retrograde Axon Transport of Herpes Simplex Virus and Pseudorabies Virus: a Live-Cell Comparative Analysis

Upon entry, neuroinvasive herpesviruses traffic from axon terminals to the nuclei of neurons resident in peripheral ganglia, where the viral DNA is deposited. A detailed analysis of herpes simplex virus type 1 (HSV-1) transport dynamics in axons following entry is currently lacking. Here, time lapse fluorescence microscopy was used to compare the postentry viral transport of two neurotropic herpesviruses: HSV-1 and pseudorabies virus (PRV). HSV-1 capsid transport dynamics were indistinguishable from those of PRV and did not differ in neurons of human, mouse, or avian origin. Simultaneous tracking of capsids and tegument proteins demonstrated that the composition of actively transporting HSV-1 is remarkably similar to that of PRV. This quantitative assessment of HSV-1 axon transport following entry demonstrates that HSV-1 and PRV share a conserved mechanism for postentry retrograde transport in axons and provides the foundation for further studies of the retrograde transport process.Herpes simplex virus type 1 (HSV-1) and the veterinary herpesvirus pathogen pseudorabies virus (PRV) establish latent infections within the peripheral nervous systems (PNS) of their hosts. Neurotropic herpesviruses gain access to the PNS at nerve endings present in infected skin or mucosal tissue. Upon entry at the nerve terminal, viral particles are transported in axons toward the neuronal cell body to ultimately deposit the viral genome into the nucleus. This process is referred to as retrograde transport and is critical for the establishment of latency. Following reactivation, progeny viral particles travel anterogradely from the ganglia toward the nerve terminals, resulting in reinfection of the dermis or other innervated tissues. Reactivated infection can manifest in various forms, including asymptomatic virus shedding or mild focal lesions (herpes labialis), or less frequently in more-severe disease (herpes keratitis, encephalitis, and in the case of varicella-zoster virus, shingles).All herpesviruses consist of an icosahedral capsid that contains the viral genome surrounded by a layer of proteins known as the tegument, which is contained within a membrane envelope (33). HSV-1 and PRV capsids disassociate from the viral envelope (2, 13, 14, 22, 23, 25, 28, 30, 40) and several tegument proteins (13, 16, 21, 25) upon fusion-mediated entry into cells. However, following entry into epithelial cell lines, the VP1/2 and UL37 tegument proteins are detected in association with cytosolic capsids of PRV by immunogold electron microscopy (16) and colocalize with HSV-1 capsids at the nuclear membrane by immunofluorescence microscopy (8). In primary sensory neurons, VP1/2 and UL37 are observed to be cotransported with PRV capsids during retrograde transport by time lapse fluorescence microscopy (21), and the kinetics of axon transport have been assessed (39).Although HSV-1 and PRV share similarities in their neurotropism in vivo (reviewed in reference 12), studies of axon transport have indicated possible mechanistic differences relevant to the underlying cell biology of neural transmission (reviewed in reference 10). As a result, a live-cell analysis comparing PRV and HSV-1 is needed to determine if axon transport mechanisms are conserved between the two neuroinvasive herpesvirus genera: Simplexvirus (HSV-1) and Varicellovirus (PRV). In this study, the retrograde transport process that delivers capsids to the nuclei of sensory neurons was compared for HSV-1 (strains KOS and F) and PRV (strain Becker).     

Propeptide-Mediated Specific Inhibition of a Recombinant Serine Protease from Indian Malaria Vector, Anopheles culicifacies

Serine proteases are a class of proteolytic enzymes that are synthesized as enzymically inactive zymogens and when required in the cell, they are activated by the removal of proregion. The role of proregions as potent and specific inhibitors of their associated protease has been established. Here, we investigated the inhibition of a recombinantly expressed and refolded Anopheles c ulicifacies serine protease (ACSP) that was isolated from the body tissue of an Indian malaria vector, A. culicifacies by its own N-terminally located 19 amino acid residue propeptide. The synthetic peptide identical to the propeptide, its three deletion mutants and leupeptin (a general serine protease inhibitor) were tested in vitro for their inhibitory activity towards recombinant ACSP. Amongst the five peptides tested, leupeptin displayed maximum inhibition closely followed by native propeptide. The reduction or loss of inhibitory potential of deletion mutants of propeptide revealed the importance of charged residues present in the propeptide for inhibition of the cognate enzyme.     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily,Cause Nagashima-type Palmoplantar Keratosis

“Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266^∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.     

Effects of Major Capsid Proteins,Capsid Assembly,and DNA Cleavage/Packaging on the pUL17/pUL25 Complex of Herpes Simplex Virus 1

The U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pU_L17/pU_L25 interaction. We found that pU_L17 and pU_L25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the U_L19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pU_L17 (i) coimmunoprecipitated with pU_L25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pU_L25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pU_L25 in the absence of the triplex protein VP23 (encoded by the U_L18 gene), (iv) required pU_L25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pU_L25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pU_L25 in infected cell nuclei required pU_L17, pU_L32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pU_L15. The data suggest that VP23 or triplexes augment the pU_L17/pU_L25 interaction and that VP23 and VP5 induce conformational changes in pU_L17 and pU_L25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pU_L17/pU_L25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.Immature herpes simplex virus (HSV) capsids, like those of all herpesviruses, consist of two protein shells. The outer shell comprises 150 hexons, each composed of six copies of VP5, and 11 pentons, each containing five copies of VP5 (23, 29, 47). One vertex of fivefold symmetry is composed of 12 copies of the protein encoded by the U_L6 gene and serves as the portal through which DNA is inserted (22, 39). The pentons and hexons are linked together by 320 triplexes composed of two copies of the U_L18 gene product, VP23, and one copy of the U_L38 gene product, VP19C (23). Each triplex arrangement has two arms contacting neighboring VP5 subunits (47). The internal shell of the capsid consists primarily of more than 1,200 copies of the scaffold protein ICP35 (VP22a) and a smaller number of protease molecules encoded by the U_L26 open reading frame, which self-cleaves to form VP24 and VP21 derived from the amino and carboxyl termini, respectively (11, 12, 19, 25; reviewed in reference 31). The outer shell is virtually identical in the three capsid types found in HSV-infected cells, termed types A, B, and C (5, 6, 7, 29, 43, 48). It is believed that all three are derived from the immature procapsid (21, 38). Type C capsids contain DNA in place of the internal shell, type B capsids contain both shells, and type A capsids consist only of the outer shell (15, 16). Cleavage of viral DNA to produce type C capsids requires not only the portal protein, but all of the major capsid proteins and the products of the U_L15, U_L17, U_L28, U_L32, and U_L33 genes (2, 4, 10, 18, 26, 28, 35, 46). Only C capsids go on to become infectious virions (27).The outer capsid shell contains minor capsid proteins encoded by the U_L25 and U_L17 open reading frames (1, 17, 20). These proteins are located on the external surface of the viral capsid (24, 36, 44) and are believed to form a heterodimer arranged as a linear structure, termed the C capsid-specific complex (CCSC), located between pentons and hexons (41). This is consistent with the observation that levels of pU_L25 are increased in C capsids as opposed to in B capsids (30). On the other hand, other studies have indicated that at least some U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) associate with all capsid types, and pU_L17 can associate with enveloped light particles, which lack capsid and capsid proteins but contain a number of viral tegument proteins (28, 36, 37). How the U_L17 and U_L25 proteins attach to capsids is not currently known, although the structure of the CCSC suggests extensive contact with triplexes (41). It is also unclear when pU_L17 and pU_L25 become incorporated into the capsid during the assembly pathway. Less pU_L25 associates with pU_L17(−) capsids, suggesting that the two proteins bind capsids either cooperatively or sequentially, although this could also be consequential to the fact that less pU_L25 associates with capsids lacking DNA (30, 36).Both pU_L25 and pU_L17 are necessary for proper nucleocapsid assembly, but their respective deletion generates different phenotypes. Deletion of pU_L17 precludes DNA packaging and induces capsid aggregation in the nuclei of infected cells, suggesting a critical early function (28, 34), whereas deletion of pU_L25 precludes correct cleavage or retention of full-length cleaved DNA within the capsid (8, 20, 32), thus suggesting a critical function later in the assembly pathway.The current studies were undertaken to determine how pU_L17 and pU_L25 associate with capsids by studying their interaction and localization in the presence and absence of other capsid proteins.     

A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar,Premolis semirufa,Is a Potent Complement System Activator

Background

The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system.

Results

The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity.

Conclusion

These data show that a serine protease present in the Premolis semirufa’s bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.     

Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPα, espPβ, espPγ, and espPδ), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPβ, espPγ, and espPδ genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPα, espPβ, espPγ, and espPδ in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPα (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPγ cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPβ and EspPδ either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

Stability of Thermostable Enzyme,Aqualysin I ; a Subtilisin-type Serine Protease from Thermus aquaticus YT-1

We characterized the heat stability and detergent stabilities of aqualysin I, produced by Thermus aquaticus YT-1, and compared them with those of fungal proteinase K and Bacillus subtilisin.

Aqualysin I displayed excellent heat and detergent stabilities. Proteinase K, another Cys-containing enzyme, was less stable than aqualysin I. All these enzymes maintained activities in the presence of urea or Tween-20.