Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

Mimicry of host chemokines and chemokine receptors to modulate chemokine activity is a strategy encoded by beta- and gammaherpesviruses, but very limited information is available on the anti-chemokine strategies encoded by alphaherpesviruses. The secretion of chemokine binding proteins (vCKBPs) has hitherto been considered a unique strategy encoded by poxviruses and gammaherpesviruses. We describe a family of novel vCKBPs in equine herpesvirus 1, bovine herpesvirus 1 and 5, and related alphaherpesviruses with no sequence similarity to chemokine receptors or other vCKBPs. We show that glycoprotein G (gG) is secreted from infected cells, binds a broad range of chemokines with high affinity and blocks chemokine activity by preventing their interaction with specific receptors. Moreover, gG also blocks chemokine binding to glycosaminoglycans, an interaction required for the correct presentation and function of chemokines in vivo. In contrast to other vCKBPs, gG may also be membrane anchored and, consistently, we show chemokine binding activity at the surface of cells expressing full-length protein. These alphaherpesvirus vCKBPs represent a novel family of proteins that bind chemokines both at the membrane and in solution.     

Equine herpesvirus 1 utilizes a novel herpesvirus entry receptor

The well-described herpesvirus entry receptors HveA (TNFRSF14), HveB (nectin 2), and HveC (nectin 1) have been shown to mediate the entry of alphaherpesviruses. Our findings showed that the alphaherpesvirus equine herpesvirus 1 (EHV-1) efficiently entered and replicated in CHO-K1 cells that lack the entry receptors HveA, HveB, and HveC, demonstrating that EHV-1 utilizes a unique entry receptor. As with other alphaherpesviruses, efficient EHV-1 entry was dependent on glycoprotein D and cell surface glycosaminoglycans.     

Herpesvirus chemokine-binding glycoprotein G (gG) efficiently inhibits neutrophil chemotaxis in vitro and in vivo

Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.     

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfp at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.     

Adaptability in herpesviruses: glycoprotein D-independent infectivity of pseudorabies virus.

Initial contact between herpesviruses and host cells is mediated by virion envelope glycoproteins which bind to cellular receptors. In several alphaherpesviruses, the nonessential glycoprotein gC has been found to interact with cell surface proteoglycans, whereas the essential glycoprotein gD is involved in stable secondary attachment. In addition, gD is necessary for penetration, which involves fusion between virion envelope and cellular cytoplasmic membrane. As opposed to other alphaherpesvirus gD homologs, pseudorabies virus (PrV) gD is not required for direct viral cell-to-cell spread. Therefore, gD- PrV can be passaged in noncomplementing cells by cocultivating infected and noninfected cells. Whereas infectivity was found to be strictly cell associated in early passages, repeated passaging resulted in the appearance of infectivity in the supernatant, finally reaching titers as high as 10(7) PFU/ml (PrV gD- Pass). Filtration experiments indicated that this infectivity was not due to the presence of infected cells, and the absence of gD was verified by Southern and Western blotting and by virus neutralization. Infection of bovine kidney cells constitutively expressing PrV gD interfered with the infectivity of wild-type PrV but did not inhibit that of PrV gD- Pass. Similar results were obtained after passaging of a second PrV mutant, PrV-376, which in addition to gD also lacks gG, gI, and gE. Penetration assays demonstrated that PrV gD- Pass entered cells much more slowly than wild-type PrV. In summary, our data demonstrate the existence of a gD-independent mode of initiation of infection in PrV and indicate that the essential function(s) that gD performs in wild-type PrV infection can be compensated for after passaging. Therefore, regarding the requirement for gD, PrV seems to be intermediate between herpes simplex virus type 1, in which gD is necessary for penetration and cell-to-cell spread, and varicella-zoster virus (VZV), which lacks a gD gene. Our data show that the relevance of an essential protein can change under selective pressure and thus demonstrate a way in which VZV could have evolved from a PrV-like ancestor.     

Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.     

Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment,penetration, growth curve and direct cell-to-cell spread

The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.     

Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment, penetration, growth curve and direct cell-to-cell spread

Glycoprotein G plays many important roles in al-phaherpesvirus except for the varicella-zoster virus (VZV), from which gG is absent[1]. The gG of most alphaherpesviruses is secreted after proteolytic proc-essing, as occurs in the herpes simplex virus 2 (HSV- 2)[2―4], bovine herpesvirus 1 (BHV-1)[5―7], bovine herpesvirus 5 (BHV-5)[8], equine herpesvirus 1 (EHV- 1)[9], equine herpesvirus 3 (EHV-3)[10], equine herpes-virus 4 (EHV-4)[11], feline herpesvirus 1 (FHV-1)[12], infectious la…     

Pseudorabies virus UL3 gene codes for a nuclear protein which is dispensable for viral replication

Many of the products of the ca. 80 genes encoded by alphaherpesviruses have already been identified and, at least tentatively, functionally characterized. Among the least characterized proteins are the products of the genes homologous to herpes simplex virus UL3, which are present only in the subfamily Alphaherpesvirinae: To identify the UL3 protein of the porcine alphaherpesvirus pseudorabies virus (PrV), the complete PrV UL3 open reading frame was cloned, expressed in Escherichia coli as a glutathione S-transferase fusion protein, and used for immunization of a rabbit. In Western blots, the generated antiserum specifically detected a 34-kDa protein in PrV-infected cells, which was absent from purified virus preparations, indicating that PrV UL3 encodes a nonstructural protein. In indirect immunofluorescence analysis, the anti-UL3 serum produced predominantly nuclear staining in transfected as well as in infected cells, which was not altered in the absence of other virus-encoded nuclear proteins such as the UL31 and UL34 gene products. To investigate UL3 function, a deletion mutant, PrV-DeltaUL3F2, was constructed and characterized. This mutant replicated and formed plaques on noncomplementing cells indistinguishable from wild-type PrV, demonstrating that PrV UL3 is not required for virus propagation in cultured cells. Moreover, ultrastructural examinations revealed no impairment of capsid formation in the nucleus, nuclear egress of capsids, virion maturation in the cytoplasm, or virus release. Thus, the overall properties of PrV UL3 are similar to those described for the homologous herpes simplex virus proteins which may be indicative of a common, yet hitherto unknown, function in alphaherpesvirus replication. However, based on our studies, an involvement of the UL3 homologs in virion formation appears unlikely.     

Structure and assembly of the RNA binding domain of bluetongue virus non-structural protein 2

Bluetongue virus non-structural protein 2 belongs to a class of highly conserved proteins found in orbiviruses of the Reoviridae family. Non-structural protein 2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. It is able to bind single-stranded RNA non-specifically, and it has been suggested that the protein is involved in the selection and condensation of the Bluetongue virus RNA segments prior to genome encapsidation. We have determined the x-ray structure of the N-terminal domain (sufficient for the RNA binding ability of non-structural protein 2) to 2.4 A resolution using anomalous scattering methods. Crystals of this apparently insoluble domain were obtained by in situ proteolysis of a soluble construct. The asymmetric unit shows two monomers related by non-crystallographic symmetry, with each monomer folded as a beta sandwich with a unique topology. The crystal structure reveals extensive monomer-monomer interactions, which explain the ability of the protein to self-assemble into large homomultimeric complexes. Of the entire surface area of the monomer, one-third is used to create the interfaces of the curved multimeric assembly observed in the x-ray structure. The structure reported here shows how the N-terminal domain would be able to bind single-stranded RNA non-specifically protecting the bound regions in a heterogeneous multimeric but not polymeric complex.     

α疱疹病毒的神经传导——轴突上的“穿梭”

α疱疹病毒经过长期的进化与宿主形成了良好的相互适应关系。其中部分α疱疹病毒具有典型的嗜神经特性,受到广泛的关注和深入的研究。嗜神经性α疱疹病毒能突破宿主屏障而感染神经元,并在其胞体内大量繁殖进而完成进一步的扩散或在胞体中建立潜伏感染。病毒无论是感染神经元还是在进一步扩散的过程中都会经历沿轴突或树突的传导过程,所以此过程是病毒生命周期中不可或缺的一部分,同时也是影响病毒入侵神经系统的关键因素。对嗜神经性α疱疹病毒在神经元内传导过程的研究不仅能深入地了解病毒,而且还能针对性地研发相应的疫苗或靶向性治疗药物,同时还可利用其神经嗜性将病毒作为解析神经环路的有力工具。文中主要对α疱疹病毒在轴突中的传导机制进行了综述,并提出病毒在轴突中传导的研究发展方向和应用价值,可为防控α疱疹病毒感染提供参考。     

In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons

The neurotropic alphaherpesviruses invade and spread in the nervous system in a directional manner between synaptically connected neurons. Until now, this property has been studied only in living animals and has not been accessible to in vitro analysis. In this study, we describe an in vitro system in which cultured peripheral nervous system neurons are separated from their neuron targets by an isolator chamber ring. Using pseudorabies virus (PRV), an alphaherpesvirus capable of transneuronal spread in neural circuits of many animals, we have recapitulated in vitro all known genetic requirements for retrograde and anterograde transneuronal spread as determined previously in vivo. We show that in vitro transneuronal spread requires intact axons and the presence of the viral proteins gE, gI, and Us9. We also show that transneuronal spread is dependent on the viral glycoprotein gB, which is required for membrane fusion, but not on gD, which is required for extracellular spread. We demonstrate ultrastructural differences between anterograde- and retrograde-traveling virions. Finally, we show live imaging of dynamic fluorescent virion components in axons and postsynaptic target neurons.     

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.     

鸭肠炎病毒UL6基因的分子特征

为了研究鸭肠炎病毒(Duck enteritis virus,DEV)UL6的特征,以DEV Clone-03株基因组DNA为模板,用靶基因步移PCR方法扩增得到UL6基因区域2534 bp的DNA片段。结果发现,DEV Clone-03 UL6基因由2373个核苷酸组成,编码一条790个氨基酸残基组成的多肽。与14株已报道的具有全UL6同源基因序列的疱疹病毒参考株进行比较,DEV Clone-03株与α疱疹病毒的核苷酸和氨基酸同源性较之与β疱疹病毒和γ疱疹病毒要高。氨基酸序列比较显示,DEV Clone-03 UL6基因具有α疱疹病毒UL6同源基因5个保守区域。UL6基因推导的氨基酸系统发育进化树分析发现,DEV Clone-03与α疱疹病毒属一个群,在α疱疹病毒中与马立克氏病病毒(Marek′sdisease herpesvirus,MDV)更接近。同时,序列分析发现,在201~222和425~441等氨基酸位,α疱疹病毒参考毒株均有不同程度的缺失,而DEV Clone-03表现出独有的完整性。     

Morphogenesis of a highly replicative EGFPVP22 recombinant Marek's disease virus in cell culture

Marek's disease virus (MDV) is an alphaherpesvirus for which infection is strictly cell associated in permissive cell culture systems. In contrast to most other alphaherpesviruses, no comprehensive ultrastructural study has been published to date describing the different stages of MDV morphogenesis. To circumvent problems linked to nonsynchronized infection and low infectivity titers, we generated a recombinant MDV expressing an enhanced green fluorescent protein fused to VP22, a major tegument protein that is not implicated in virion morphogenesis. Growth of this recombinant virus in cell culture was decreased threefold compared to that of the parental Bac20 virus, but this mutant was still highly replicative. The recombinant virus allowed us to select infected cells by cell-sorting cytometry at late stages of infection for subsequent transmission electron microscopy analysis. Under these conditions, all of the stages of assembly and virion morphogenesis could be observed except extracellular enveloped virions, even at the cell surface. We observed 10-fold fewer naked cytoplasmic capsids than nuclear capsids, and intracellular enveloped virions were very rare. The partial envelopment of capsids in the cytoplasm supports the hypothesis of the acquisition of the final envelope in this cellular compartment. We demonstrate for the first time that, compared to other alphaherpesviruses, MDV seems deficient in three crucial steps of viral morphogenesis, i.e., release from the nucleus, secondary envelopment, and the exocytosis process. The discrepancy between the efficiency with which this MDV mutant spreads in cell culture and the relatively inefficient process of its envelopment and virion release raises the question of the MDV cell-to-cell spreading mechanism.     

A two-stage model for lipid modulation of the activity of integral membrane proteins

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes.     

Solution structure and characterization of the DNA-binding activity of the B3BP-Smr domain

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer α-β sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.     

The pros and cons of autophagic flux among herpesviruses

Autophagy has been intensively studied in herpes simplex virus type 1 (HSV-1), a human alphaherpesvirus. The HSV-1 genome encodes a well-known neurovirulence protein called ICP34.5. When the gene encoding this protein is deleted from the genome, the virus is markedly less virulent when injected into the brains of animal models. Subsequent characterization of ICP34.5 established that the neurovirulence protein interacts with BECN1, thereby inhibiting autophagy and facilitating viral replication in the brain. However, an ortholog of the ICP34.5 gene is lacking in the genomes of other closely related alphaherpesviruses, such as varicella-zoster virus (VZV). Further, autophagosomes are easily identified in the exanthem (rash) that is the hallmark of both VZV diseases—varicella and herpes zoster. Inhibition of autophagy leads to diminished VZV titers. Finally, no block is detected in studies of autophagic flux following VZV infection. Thus autophagy appears to be proviral during VZV infection while antiviral during HSV-1 infection. Because divergence to this degree is extremely unusual for 2 closely related herpesviruses, we postulate that VZV has accommodated its infectious cycle to benefit from autophagic flux, whereas HSV-1 has captured cellular immunomodulatory genes to inhibit autophagy.