Varicella-zoster Virus (VZV) Mediates a Delayed Host ShutoffIndependent of Open Reading Frame (ORF) 17 Expression

Varicella-zoster virus (VZV) open reading frame 17 (ORF 17) is the gene corresponding to Herpes simplex-virus (HSV) UL41. The UL41 gene encodes the virion host shutoff factor (vhs), a RNase that has been the object of detailed studies. In contrast to HSV, knowledge about VZV mediated shutoff effects and the role of ORF 17 is poor.We investigated the ORF 17 expression in infected cells and analyzed shutoff effects. ORF 17 expression could not be proven in infected human fibroblast cell lines and melanoma (MeWo) cells. Only after induction by Phorbol 12-myristate 13-acetate an ORF 17 expression became detectable in MeWo cells. Nevertheless, using stable expressed GAPDH mRNA as a marker for mRNA degradation, a VZV mediated shutoff, independent of ORF 17 expression, became measurable. Transfection experiments demonstrated that transient ORF 17 expression did not decrease the cellular GAPDH mRNA level. We examined whether the VZV shutoff factor is a tegument protein causing an early shutoff or whether it needs to be expressed (delayed shutoff). The GAPDH mRNA level in Actinomycin D pretreated and infected MeWo cells did not decrease even faster than the theoretical decay rate based on a half-life of 24 h. These findings lead to the conclusion that the VZV shutoff factor is not a mature protein localized in the virion and that VZV causes a delayed virion host shutoff effect.     

Identification of structural proteins of koi herpesvirus

As a prerequisite for development of improved vaccines and diagnostic tools for control of the fish pathogen koi herpesvirus, or cyprinid herpesvirus 3 (CyHV-3), we have started to identify putative viral envelope and capsid proteins. The complete or partial CyHV-3 open reading frames ORF25, ORF65, ORF92, ORF99, ORF136, ORF138, ORF146, ORF148, and ORF149 were expressed as bacterial fusion proteins, which were then used for preparation of monospecific rabbit antisera. All of the sera that were obtained detected their target proteins in cells transfected with the corresponding eukaryotic expression plasmids. However, only the type I membrane proteins pORF25, pORF65, pORF99, pORF136 and pORF149 and the major capsid protein pORF92 were sufficiently abundant and immunogenic to permit unambiguous detection in CyHV-3-infected cells. In indirect immunofluorescence tests (IIFT), sera from naturally or experimentally CyHV-3-infected carp and koi predominantly reacted with cells transfected with expression plasmids encoding pORF25, pORF65, pORF148, and pORF149, which represent a family of related CyHV-3 membrane proteins. Moreover, several neutralizing monoclonal antibodies raised against CyHV-3 virions proved to be specific for pORF149 in IIFT of transfected cells and in immunoelectron microscopic analysis of CyHV-3 particles. Since pORF149 appears to be an immunorelevant envelope protein of CyHV-3, a recombinant baculovirus was generated for its expression in insect cells, and pORF149 was shown to be incorporated into pseudotyped baculovirus particles, which might be suitable as diagnostic tools or subunit vaccines.     

Cloning and sequence analysis of a Marek's disease virus origin binding protein (OBP) reveals strict conservation of structural motifs among OBPs of divergent alphaherpesviruses

Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. In its natural host, MDV induces Marek's disease (MD), a lethal condition characterized by malignant lymphoma of T cells. Although symptoms of MD may be prevented by vaccination, no practical pharmacological method of control has been widely accepted. Viral replication represents a point at which pharmacological control of herpesvirus infection may be most successful. However, this requires detailed knowledge of viral replication proteins. Studies in HSV-1 DNA replication implicate the UL9 protein as a key initiator of replication. For example, binding of UL9 to HSV-1 origins is a prerequisite for assembly of additional replication proteins. In this study, a protein, whose apparent molecular size is similar to that of HSV-1 UL9, was identified in extracts of MDV infected cells by western blot analysis with anti-HSV-1 UL9 antibody. A putative MDV UL9 gene was subsequently identified through sequencing of MDV genome fragments (BamHI G and C). Extended DNA sequence analysis revealed an open reading frame (ORF) which could encode a protein homologous to HSV-1 UL9. The MDV UL9 ORF encodes 841 amino acids, producing a sequence 49% identical to HSV-1 UL9 and 46% identical to VZV gene 51 product (VZV UL9). MDV UL9 shares numerous structural motifs with HSV-1 and VZV UL9 proteins, including six conserved N-terminal helicase motifs, an N-terminal leucine zipper motif, a C-terminal pseudo-leucine zipper sequence, and a putative helix-turn-helix structure.     

A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus

Summary.  Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M_r) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP₃) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP₃ protein was present in amounts equivalent to those of the N, M, and GP₅ proteins in the infected cells, whereas no GP₃ could be detected in purified virions. During the first 30 min of chase, the GP₃ undergoes a gradual downward shift of its apparent M_r, thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural. Received February 3, 1998 Accepted May 16, 1998     

Amino-terminal epitopes are exposed when full-length open reading frame 2 of hepatitis E virus is expressed in Escherichia coli,but carboxy-terminal epitopes are masked

We constructed a panel of overlapping and non-overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S-transferase (GST), from which proteins were expressed in Eschericia coli. IgG-specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques (Macaca mulatta) or from HEV-infected patients. Under these conditions, full-length ORF2 protein (GST-ORF2) was strongly reactive with acute-phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST-ORF2.3, representing amino acids 1–110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full-length protein. Conversely, GST-ORF2.1, representing amino acids 394–660 of the ORF2 protein was strongly reactive with both acute- and convalescent-phase sera. Extension of GST-ORF2.1 towards the N-terminus led to a progressive loss of convalescent-phase reactivity, apparent with as few as 20 additional HEV-specific amino acids. Deletion of 40 or more amino acids from the N-terminus of ORF2.1 also led to reduced convalescent-phase reactivity, however a protein representing this “reactive” region, containing amino acids 394–473, was poorly reactive, suggesting that the convalescent-reactive epitopes are conformational. Expression of full-length ORF2 protein in E. coli therefore masks the convalescent-reactive epitopes within the C-terminal part of the protein, without affecting N-terminal, acute-reactive epitopes. J. Med. Virol. 52:289–300, 1997. © 1997 Wiley-Liss, Inc.     

The Varicella-zoster virus ORF54 gene product encodes the capsid portal protein, pORF54

The Varicella-zoster virus (VZV) ORF54 gene was characterized using a guinea pig antiserum prepared to a GST-pORF54 fusion protein. A protein of the predicted size, 87kDa, was detected in VZV-infected MeWo cells but not in mock-infected cells. Sucrose density gradient fractionation of pORF54 expressed in a recombinant baculovirus system resulted in samples containing enriched amounts of pORF54. Electron microscopic analysis suggested that the ORF54 gene encodes a protein that assembles into ring-like portal structures similar to those observed for numerous bacteriophages and other herpesviruses.     

Interference of porcine circovirus type 2 ORF2 immunogenicity by ORF1 and ORF3 mixed DNA immunizations in mice

Little is known about the influences of other porcine circovirus type 2 (PCV2) proteins on the immunogenicity of Cap protein. Here we constructed plasmids expressing the ORF1 (pORF1) and ORF3 (pORF3) of PCV2, and mixed either of them with the plasmid expressing ORF2 (pORF2) as combined DNA vaccines, to compare their immunogenicity and protective efficacy. Our data revealed that pORF1 reduced the Cap-specific CD8⁺cell frequency, and both pORF1 and pORF3 attenuated the Cap-specific Th1 and post-challenge-recall VN antibody responses induced by the pORF2 plasmid, despite successful induction of Rep and ORF3 antibodies by pORF1 and pORF3, respectively. Subsequently, protocols with pORF1 or pORF3 showed significantly decreased protective efficacy compared to pORF2 alone. Overall, our data suggested that the ORF1- and ORF3-encoded Rep and ORF3 proteins may interfere with the cellular, humoral and protective immunity of the ORF2-encoded Cap protein in vivo.     

Varicella-zoster virus open reading frame 47 (ORF47) protein is critical for virus replication in dendritic cells and for spread to other cells

Varicella-zoster virus infects human dendritic cells (DCs). We found that VZV infection of DCs resulted in down-regulation of Fas expression on the surface of cells. VZV ORF47 was critical for replication of virus in human immature, but not mature DCs. Immature DCs infected with a mutant virus unable to express ORF47 expressed similar levels of a VZV immediate-early protein as cells infected with parental virus; however, cells infected with the ORF47 mutant expressed lower levels of glycoprotein E. Thus, in the absence of ORF47 protein, there is a block in viral replication between immediate-early and late gene expression. VZV unable to express ORF47 was severely impaired for spread of virus from DCs to melanoma cells. Infection of DCs with parental VZV resulted in a different pattern of phosphoproteins compared with the ORF47 mutant virus. Thus, VZV ORF47 is important for replication in immature DCs and for spread to other cells.     

Association of Two Membrane Proteins Encoded by Herpes Simplex Virus Type 2, UL11 and UL56

Herpes simplex virus (HSV) acquires envelope by budding into trans-Golgi network (TGN)-derived vesicles. Previous studies showed that the UL11 gene product enables efficient virion envelopment and export from infected cells and is incorporated into virions as tegument protein. At its N-terminus, UL11 is dually acylated by myristoic and palmitoic acids. Fatty acylation of UL11 provides both membrane binding strength and Golgi-targeting specificity. We show here that UL11 interacts with UL56 protein, a tail-anchored type II membrane protein encoded by HSV, which associated with the Golgi apparatus and cytoplasmic vesicles. We previously showed that UL56 is involved in vesicular transport in infected cells. The UL11–UL56 complex localized to the perinuclear region of the cytoplasm in infected cells. Fatty acylation of UL11 was important for the formation of the UL11–UL56 protein complex. Taken together, our results identify a novel interaction between two HSV proteins facilitated by mutual interactions with Golgi-derived vesicles.     

Varicella-zoster virus (VZV) ORF65 virion protein is dispensable for replication in cell culture and is phosphorylated by casein kinase II, but not by the VZV protein kinases

The unique short region of varicella zoster virus (VZV) encodes four genes. One of these, ORF65, is predicted to encode an 11-kDa protein. Antibody to ORF65 protein immunoprecipitated a 16-kDa protein from the membrane fraction of VZV-infected cells. ORF65 protein was shown to be phosphorylated by casein kinase II. The VZV ORF47 or ORF66 protein kinases were not required for phosphorylation of ORF65. VZV with a large deletion in ORF65 was constructed and was shown to be dispensable for replication of virus in cell culture. The herpes simplex virus homolog of VZV ORF65 has been reported to be located in the nucleus of infected cells and in virions as a tegument protein, whereas the pseudorabies virus homolog is located in the Golgi apparatus of infected cells and in virions as a type II membrane protein. The ORF65 protein localized to the Golgi apparatus in virus-infected cells and was located in virions, most likely as a type II membrane protein. Thus, VZV ORF65 more closely resembles its pseudorabies virus homolog in its localization in infected cells and virions.     

Varicella-zoster virus ORF1 gene product is a tail-anchored membrane protein localized to plasma membrane and trans-Golgi network in infected cells

Varicella-zoster virus (VZV) encodes five genes that do not have herpes simplex virus homologs. One of these genes, VZV open reading frame 1 (ORF1), encodes a membrane protein with a hydrophobic domain at its C-terminus that is predicted to be the transmembrane domain. However, the detailed characterization of ORF1 protein in infected cells has not been reported. Here, we produced mono-specific antibodies against ORF1 protein and characterized the gene products in infected cells. Western blot analyses showed the ORF1 polypeptides had apparent molecular masses of approximately 14-17 kDa. Furthermore, ORF1 was found to be a phosphoprotein by immunoprecipitation assay. In immunofluorescence assays, the VZV ORF1 protein was detected at both the plasma membrane and trans-Golgi network in both VZV-infected and ORF1-transfected cells. Moreover, ORF1 proteins associated with each other to form homodimer, and were incorporated into viral particles. The C-terminal hydrophobic domain was required for the association of ORF1 with the membrane structures, indicating that ORF1 protein is anchored to the membrane thorough its C-terminus, which is a transmembrane domain. Because ORF1 possesses a C-terminal transmembrane domain without an N-terminal signal sequence for its translocation to the ER lumen, ORF1 can be classified as a tail-anchored membrane protein. These results show that the N terminus of ORF1 protein faces the cytoplasm in infected cells and the tegument region in mature virions.     

Memory cytotoxic T cell responses to viral tegument and regulatory proteins encoded by open reading frames 4, 10, 29, and 62 of varicella-zoster virus

Cytotoxic T cell recognition of tegument and regulatory proteins encoded by open reading frames (ORFs) 4, 10, 29, and 62 of varicella-zoster virus (VZV) was evaluated using limiting dilution conditions to estimate the precursor frequencies of memory T cells specific for these proteins in immune subjects. Responder cell frequencies for ORFs 4, 10, and 62 gene products, which are virion tegument components and function as immediate early viral transactivating proteins, were equivalent. CTLp recognition of VZV proteins made in latently infected cells, which include ORF4 and ORF62 proteins, was not maintained preferentially when compared to ORF10 protein, which has not been shown to be expressed during latency. T cell recognition of ORF29 protein, the major DNA binding protein, which is expressed during replication but not incorporated into the virion tegument, was less common than responses to ORFs 4, 10, and 62 gene products. Older individuals had diminished numbers of memory CTLp that lysed autologous targets expressing IE62 protein; these responses were increased after immunization with live attenuated varicella vaccine to the range observed in younger adults. Adaptive immunity to VZV is characterized by a broad repertoire of memory CTL responses to proteins that comprise the virion tegument and regulate viral gene expression in infected cells.     

Intracellular localization of varicella-zoster virus ORF39 protein and its functional relationship to glycoprotein K

Varicella-zoster virus (VZV) encodes two multiply inserted membrane proteins, open reading frame (ORF) 39 protein (ORF39p) and glycoprotein K (gK). The HSV-1 homologs of these proteins are believed to act in conjunction with each other during viral egress and cell-cell fusion, and they directly influence each other's intracellular trafficking. However, ORF39p and VZV gK have received very limited study largely due to difficulties in producing antibodies to these highly hydrophobic proteins. To overcome this obstacle, we introduced epitope tags into both ORF39p and gK and examined their intracellular distributions in transfected and infected cells. Our data demonstrate that both ORF39p and gK accumulate predominately in the ER of cultured cells when expressed in the absence of other VZV proteins or when coexpressed in isolation from other VZV proteins. Therefore, the transport of VZV ORF39p and gK does not exhibit the functional interdependence seen in their HSV-1 homologs. However, during infection, the primary distributions of ORF39p and gK shift from the ER to the Golgi, and they are also found in the plasma membrane indicating that their intracellular trafficking during infection depends on other VZV-encoded proteins. During infection, ORF39p and gK tightly colocalize with VZV envelope glycoproteins B, E and H; however, the coexpression of ORF39p or gK with other individual viral glycoproteins is insufficient to alter the transport of either ORF39p or gK.     

Varicella-zoster virus assembly protein p32/p36 is present in DNA-containing as well as immature capsids

Varicella-zoster virus (VZV) produces a group of nucleocapsid proteins (the p32/p36 nucleoprotein complex) which are the VZV analogues of the herpes simplex virus type 1 (HSV-1) and cy0tomegalovirus (CMV) assembly proteins. There are multiple components in the VZV p32/p36 complex, with major proteins of 32 and 36 kDa and minor proteins of 34 and 38 kDa. In HSV-1 the assembly proteins have been shown to be present in immature (B) capsids, but are removed prior to the formation of mature (C) capsids containing the viral DNA genome. Our work has shown that VZV produces capsids corresponding to the B and C forms. However, in contrast to HSV-1, VZV also produces “B/C” capsids that appear to contain both the assembly proteins and the viral DNA genome. Possible mechanisms for this are discussed. In addition, it was shown that VZV capsids appear to lack the 36 and 38 kDa proteins, and based on this observation we suggest that these may represent unprocessed forms of the assembly protein. In both HSV and CMV, a much larger, crossreactive protein has been identified as the fulllength product of the gene coding for the assembly protein. The homologous VZV gene (ORF 33) theoretically has the capacity to produce a 66 kDa protein. However, no such protein is readily apparent in VZV-infected cells. The presence of an immunoreactive 64 kDa protein was demonstrated in purified VZV capsids which may represent the full-length ORF 33 protein. © 1995 Wiley-Liss, Inc.     

The role of protein kinase activity expressed by the UL13 gene of herpes simplex virus 1: the activity is not essential for optimal expression of UL41 and ICP0

Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that is packaged into virions and regulates optimal expression of ICP0 and a subset of late (gamma) proteins, including UL41 in infected cells. In the present study, we investigated the role(s) of the protein kinase activity of UL13 in viral replication using a recombinant virus expressing enzymatically inactive UL13 after an amino acid substitution in the invariant lysine of UL13. The recombinant virus carrying this mutation formed smaller plaques yielded 10-fold less progeny than wild-type virus but could not be differentiated from wild-type virus with respect to accumulation of UL41 and ICP0 in infected cells. These results indicate that the protein kinase activity of UL13 plays a role in viral replication in cell culture, but the activity is not essential for the optimal expression of UL41 and ICP0.     

Cloning,expression, and immunogenicity of the assembly protein of varicella-zoster virus and detection of the products of open reading frame 33

Herpesviruses produce assembly proteins (AP) that act as scaffolding proteins for the assembly of the viral capsids. The products of the assemblin gene, which encodes both maturational protease and AP, have been established for herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (CMV). We cloned an inframe ORF (encoding amino acids 304–605), found within the ORF 33 assemblin gene of VZV, into a yeast expression vector. The 34-kDa AP was expressed as a fusion protein with the particle-forming Ty p1 protein, resulting in high-level production of hybrid AP-virus-like particles (AP-VLPs). When AP-VLPs were injected into mice and rabbits, antibodies were produced that reacted with, but that did not neutralise, native VZV. Three of four inbred strains of mice immunised with AP-VLPs produced a VZV-specific T-cell response. The mouse and rabbit sera reacted with six bands on native VZV by Western blot analysis. The dominant bands were found at 34 and 38 kDa. Bands were also seen at 66, 63, 41, and 31 kDa. The 38-kDa protein may represent the mature AP derived from the 41-kDa precursor AP, itself the release product from the full-length 66-kDa assemblin. The 34-kDa protein probably represents the product of the inframe co-translational gene within ORF 33 encoding amino acids 304–605. The genetic organisation and proteolytic maturation of VZV assemblin are, therefore, analogous to those of other herpesviruses. J. Med. Virol. 53:332–339, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutant proteins: correlation with in vivo function

The DNA polymerase (pol) catalytic subunit of herpes simplex virus type 1, encoded by UL30, and its accessory factor, UL42 protein, are both essential for the replication of the virus. Because the stable interaction between UL42 and pol renders the pol fully processive for replicative DNA synthesis, disruption of this interaction represents a potential goal in the development of novel antiviral compounds. To better compare the effects of mutations in UL42 protein on its known in vitro functions, mutations were expressed as glutathione-S-transferase (GST)-fusions and the fusion proteins used in affinity chromatography. In this report, we demonstrate the relationship between the abilities of mutant UL42 fusion proteins to bind pol and to stimulate pol activity in vitro, and the abilities of nonfusion mutant proteins to function in viral replication. The pol stimulation assay using GST fusion proteins was found to be a more accurate and sensitive measure of the ability of the UL42 protein to function in vitro than the pol binding assay using the fusion proteins linked to a solid matrix. We also found an excellent correlation between the ability of purified GST fusion proteins to stimulate pol activity in vitro and the ability of full-length nonfusion UL42 mutant genes to support DNA replication in infected cells. Our results demonstrate that two noncontiguous stretches of amino acids, from 137 to 142 and from 274 to 282, are essential for UL42 function in vivo and in vitro. Although mutant d241-261 exhibited close to wild-type abilities to stimulate pol activity in vitro, it was not capable of complementing the replication of a UL42 null mutant virus. The region of UL42 protein within or close to 241-261 may serve to hinge the essential regions within the N- and C-terminal portions of the protein which are thought to interdigitate. It is hypothesized that reduction in the length of the hinge region could alter the ability of UL42, and/or its complex with pol, to function with one or more of the other proteins present in the DNA replisome within infected cells.     

pUL25 immunolocalization in human cytomegalovirus-infected and gene-transfected cells

Summary.  By means of confocal and electron microscope immunocytochemistry we have studied the localization of a recently described structural protein (pUL25) of human cytomegalovirus, in both infected cells and in cells transiently transfected with UL25. pUL25 localization in infected cells was observed in typical cytoplasmic structures characterized by a very electrondense texture previously reported to accumulate other tegument proteins. At the virion level pUL25 seems to localize at the interface between the tegument and the capsid of both intracytoplasmic and extracellular virions. In UL-25-transfected cells, pUL25 has been found in characteristic para-crystalline cytoplasmic aggregates, suggesting its intrinsic ability to aggregate in a regular subunit pattern. Received August 6, 1999/Accepted November 3, 1999