Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction.

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.

Vaccinia virus-free recovery of vesicular stomatitis virus.

The advent of reverse-genetics represents a powerful new approach to elucidate aspects of negative-sense RNA virus replication. The reverse-genetics system established previously for vesicular stomatitis virus (VSV) required four plasmids encoding the nucleoprotein (N), phosphoprotein (P), polymerase (L), and the full-length, anti-genomic RNA. Transcription to yield the antigenomic RNA as well as the N, P, and L, mRNAs was initiated by bacteriophage T7 polymerase expressed from a recombinant Vaccinia virus. In this report, we describe the successful recovery of infectious VSV in the absence of Vaccinia virus. The N, P, and L genes of VSV were inserted downstream of both the T7 promoter and an internal ribosomal entry site (IRES element). T7 polymerase was expressed constitutively from BSR-T7/5 cells. RTPCR was used to confirm that the recovered VSV was derived from transfected DNA. Virion protein profile, CPE in tissue culture, and virus titer of the recombinant VSV were indistinguishable from those of parental VSV. Thus, the need for Vaccinia virus is eliminated with this system, making it an attractive, alternative approach for the recovery of infectious VSV from DNA.

A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding.

VP40, the putative matrix protein of both Ebola and Marburg viruses, possesses a conserved proline-rich motif (PY motif) at its N terminus. We demonstrate that the VP40 protein can mediate its own release from mammalian cells, and that the PY motif is important for this self-exocytosis (budding) function. In addition, we used Western-ligand blotting to demonstrate that the PY motif of VP40 can mediate interactions with specific cellular proteins that have type I WW-domains, including the mammalian ubiquitin ligase, Nedd4. Single point mutations that disrupted the PY motif of VP40 abolished the PY/WW-domain interactions. Significantly, the full-length VP40 protein was shown to interact both physically and functionally with full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4. The VP40 protein was multiubiquitinated by Rsp5 in a PY-dependent manner in an in vitro ubiquitination assay. These data demonstrate that the VP40 protein of Ebola virus possesses a PY motif that is functionally similar to those described previously for Gag and M proteins of specific retroviruses and rhabdoviruses, respectively. Last, these studies imply that VP40 likely plays an important role in filovirus budding, and that budding of retroviruses, rhabdoviruses, and filoviruses may proceed via analogous mechanisms.

WW- and SH3-domain interactions with Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection.

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.

A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding.

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the gag-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.

Late domain function identified in the vesicular stomatitis virus M protein by use of rhabdovirus-retrovirus chimeras.

Little is known about the mechanisms used by enveloped viruses to separate themselves from the cell surface at the final step of budding. However, small sequences in the Gag proteins of several retroviruses (L domains) have been implicated in this process. A sequence has been identified in the M proteins of rhabdoviruses that closely resembles the PPPPY motif in the L domain of Rous sarcoma virus (RSV), an avian retrovirus. To evaluate whether the PPPY sequence in vesicular stomatitis virus (VSV) M protein has an activity analogous to that of the retroviral sequence, M-Gag chimeras were characterized. The N-terminal 74 amino acids of the VSV (Indiana) M protein, including the PPPY motif, was able to replace the L domain of RSV Gag and allow the assembly and release of virus-like particles. Alanine substitutions in the VSV PPPY motif severely compromised the budding activity of this hybrid protein but not that of another chimera which also contained the RSV PPPPY sequence. We conclude that this VSV sequence is functionally homologous to the RSV L domain in promoting virus particle release, making this the first example of such an activity in a virus other than a retrovirus. Both the RSV and VSV motifs have been shown to interact in vitro with certain cellular proteins that contain a WW interaction module, suggesting that the L domains are sites of interaction with unknown host machinery involved in virus release.

A chimeric influenza virus expressing an epitope of outer membrane protein F of Pseudomonas aeruginosa affords protection against challenge with P. aeruginosa in a murine model of chronic pulmonary infection.

The ability of a chimeric influenza virus containing, within the antigenic B site of its hemagglutinin, an 11-amino-acid (AEGRAINRRVE) insert from the peptide 10 epitope of outer membrane (OM) protein F of Pseudomonas aeruginosa to serve as a protective vaccine against P. aeruginosa was tested by using the murine chronic pulmonary infection model. Mice immunized with the chimeric virus developed antibodies that reacted in an enzyme-linked immunosorbent assay with peptide 10, with purified protein F, and with whole cells of various immunotype strains of P. aeruginosa but failed to react with a protein F-deficient strain of P. aeruginosa. The chimeric-virus antisera reacted specifically with protein F alone when immunoblotted against proteins extracted from cell envelopes of each of the seven Fisher-Devlin immunotype strains and had significantly greater in vitro opsonic activity for P. aeruginosa than did antisera from wild-type influenza virus-immunized mice. Subsequent to intratracheal challenge with agar-encased cells of P. aeruginosa, chimeric-virus-immunized mice developed significantly fewer severe lung lesions than did control mice immunized with the wild-type influenza virus. Furthermore, the chimeric influenza virus-immunized group had a significantly smaller percentage of mice with >5 x 10(3) CFU of P. aeruginosa in their lungs upon bacterial quantitation than did the control group. These data indicate that chimeric influenza viruses expressing epitopes of OM protein F warrant continued development as vaccines to prevent pulmonary infections caused by P. aeruginosa.

Chimeric influenza viruses incorporating epitopes of outer membrane protein F as a vaccine against pulmonary infection with Pseudomonas aeruginosa.

Peptide 10 (NATAEGRAINRRVE, residues 305-318 of mature protein F) is one of two linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa both of which have been shown to elicit whole cell-reactive antibodies and to afford protection in animal models against P. aeruginosa infection. Influenza A virus was chosen as a vector to present this epitope in a human-compatible vaccine. Various lengths of the peptide 10 epitope ranging from a 5-mer (GRAIN), 7-mer (AINRRVE), 8-mer (TAEGRAIN), 9-mer (GRAINRRVE), 11-mer (AEGRAINRRVE) to a 12-mer (TAEGRAINRRVE) were attempted to be presented into the antigenic B-site of the hemagglutinin (HA) of live recombinant influenza virus. Using PCR, DNA sequences encoding these various peptide 10 lengths were inserted into the HA gene of influenza A/WSN/33 virus. By using a reverse-genetics transfection system, RNA transcribed in vitro from these chimeric HA genes was reassorted into infectious virus. To date chimeric viruses have been rescued and purified containing the peptide 10 5-mer, 7-mer, 8-mer, and 11-mer. RT-PCR and sequencing have confirmed the presence of P. aeruginosa sequences in the HA RNA segment of each chimeric virus. Each of the four chimeric viruses produced to date was used to immunize mice to determine the ability of each chimeric virus to elicit antibodies reactive with whole cells of P. aeruginosa. The immunization protocol consisted of a series of three intranasal inoculations, followed by two intramuscular injections of the chimeric virus. The chimeric virus incorporating the 11-mer elicited IgG antibodies that reacted with various immunotype strains of P. aeruginosa in a whole cell ELISA at titers of 80 to 2,560, whereas the chimeric virus incorporating the 8-mer elicited whole cell-reactive IgG antibodies at titers of 320 to 2,560. These data suggest that these two chimeric viruses may have vaccine efficacy against P. aeruginosa infection. These studies may result in the development of a chimeric influenza virus-protein F vaccine which would prove to be suitable for use in children with cystic fibrosis for the prevention of pulmonary colonization of these children with P. aeruginosa.

Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection.

Defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) are capable of mediating persistent infection (S. A. Dauenhauer, R. A. Robinson, and D. J. O'Callaghan, J. Gen. Virol. 60:1-14, 1982; R. A. Robinson, R. B. Vance, and D. J. O'Callaghan, J. Virol. 36:204-219, 1980). Sequence analysis of cloned DI particle DNA revealed that portions of two regulatory genes, ICP22 (IR4) and ICP27 (UL3), are linked in frame to form a unique hybrid open reading frame (ORF). This hybrid ORF, designated as the IR4/UL3 gene, encodes the amino-terminal 196 amino acids of the IR4 protein (ICP22 homolog) and the carboxy-terminal 68 amino acids of the UL3 protein (ICP27 homolog). Portions of DNA sequences encoding these two regulatory proteins, separated by more than 115 kbp in the standard virus genome, were linked presumably by a homologous recombination event between two identical 8-bp sequences. Reverse transcriptase-PCR and S1 nuclease analyses revealed that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the ICP22 and ICP27 proteins demonstrated that a 31-kDa hybrid protein was synthesized in the DI particle-enriched infection but not in standard virus infection. This 31-kDa hybrid protein was expressed at the same time as the ICP22 protein in DI particle-enriched infection and migrated at the same location on polyacrylamide gel electrophoresis as the protein expressed from a cloned IR4/UL3 expression vector. These observations suggested that the unique IR4/UL3 hybrid gene is expressed from the DI particle genome and may play a role in DI particle-mediated persistent infection.

Measles virus phosphoprotein (P) requires the NH2- and COOH-terminal domains for interactions with the nucleoprotein (N) but only the COOH terminus for interactions with itself.

A mammalian two-hybrid system was used to characterize protein-protein interactions between the measles virus nucleoprotein (N) and phosphoprotein (P). Progressive deletions at both the amino- and carboxy-termini of P facilitated the mapping of two distinct domains on P that are important for interaction with N: (i) a domain mapping predominantly within the C-terminal 100 amino acids and (ii) a domain composed of the extreme amino-terminal residues. Using the same two-hybrid assay, we discovered that the P protein interacts strongly with itself. In contrast to the N-P interaction, only a single C-proximal domain of P was essential for P-P interaction.

Mutations within noncoding terminal sequences of model RNAs of Sendai virus: influence on reporter gene expression.

A reverse-genetics system employing the chloramphenicol acetyltransferase (CAT) reporter gene has been established previously for Sendai virus. We utilized PCR-directed mutagenesis to introduce nucleotide additions, deletions, and/or substitutions within terminal Sendai virus RNA sequences. The influence of these mutations on replication-transcription of these model Sendai-CAT RNAs was analyzed by assaying CAT activity. Results from these experiments indicate that (i) Sendai-CAT RNAs expressing wild-type levels of CAT activity conform to the Sendai virus rule of six, (ii) apparent exceptions to the rule of six exist in that the 5' terminus of the Sendai-CAT RNA is more tolerant than the 3' terminus of nucleotide additions or deletions, and (iii) the 3' leader region of Sendai-CAT RNA appears to be sensitive not only to mutagenesis (single-nucleotide addition or deletion) but also to changes in its total nucleotide length.

Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies.

During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro-translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.

Identification and characterization of the ICP22 protein of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.

Characterization of the myristylated polypeptide encoded by the UL1 gene that is conserved in the genome of defective interfering particles of equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The UL1 gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. UL1 has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 UL1 gene belongs to the early kinetic class and is transcribed as a 1.2-kb polyadenylated mRNA (R. N. Harty, R. R. Yalamanchili, and D. J. O'Callaghan, Virology 183:830-833, 1991). In this report, the UL1 protein was identified and characterized as a 33-kDa polypeptide in EHV-1-infected cells by using rabbit polyclonal antiserum raised against a TrpE-UL1 fusion protein (amino acids 7 to 258 of UL1) synthesized in Escherichia coli. Results from Western blot (immunoblot), immunoprecipitation, indirect immunofluorescence, and biochemical analyses indicated that the UL1 polypeptide (i) is more abundant in cells infected with DIP-enriched virus than in cells infected with standard EHV-1, (ii) is synthesized as early as 3 h postinfection (p.i.) in infection with standard virus or in infection with DIP-enriched virus preparations and increases in abundance up to 12 h p.i., (iii) appears to be associated with the rough endoplasmic reticulum-Golgi apparatus early in infection (3 to 4 h p.i.), while a diffuse cytoplasmic pattern of fluorescence is observed late in infection (7 to 8 h p.i.), (iv) is modified by myristic acid as it contains a consensus N-terminal myristylation site and is readily labeled with [3H]myristic acid, and (v) is associated with mature EHV-1 virions.

Transcriptional and translational analyses of the UL2 gene of equine herpesvirus 1: a homolog of UL55 of herpes simplex virus type 1 that is maintained in the genome of defective interfering particles.

Defective interfering particles (DIPs) of equine herpesvirus 1 (EHV-1; Kentucky A strain) mediate persistent infection. DNA sequences at the L terminus, which contain the UL2 gene (homolog of UL55 of herpes simplex virus type 1 and open reading frame 3 of varicella-zoster virus) of standard EHV-1, have been shown to be highly conserved in all clones of the EHV-1 DIP genome. The UL2 mRNA was characterized by S1 nuclease analyses, which mapped the 5' and 3' termini of the 0.9-kb early UL2 mRNA to approximately 26 and 16 nucleotides downstream of a TTTAAA box and polyadenylation signal, respectively. The UL2 open reading frame, present within both the EHV-1 standard and DIP genomes, was inserted into the transcription expression vector pGEM-3Z to yield constructs pGEML2 and pDIL2, respectively. After in vitro transcription and translation, both constructs yielded a comigrating 23-kDa protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal antiserum was raised against the UL2 protein by injecting rabbits with a TrpE/UL2 fusion protein expressed from plasmid pATH23L2 in Escherichia coli. The UL2-specific antiserum reacted in Western immunoblot and immunoprecipitation analyses with a 23-kDa polypeptide synthesized in cells infected with standard EHV-1 or DIP-enriched virus. These data also indicated that the UL2 polypeptide was more abundant in DIP-infected cells than in standard EHV-1-infected cells. Results from time course and pulse-chase analyses suggested that the UL2 polypeptide has a rapid turnover rate in DIP-infected cells.

Identification and expression of the UL1 gene product of equine herpesvirus 1.

Sequences encoding the UL1 gene of equine herpesvirus type 1 (EHV-1) are conserved in the genome of EHV-1 defective interfering particles (DIPs) that mediate oncogenic transformation and persistent infection. The UL1 protein was identified by in vitro transcription/translation and hybrid-arrest translation analyses which employed a UL1/pGEM-3Z construct designated pGEML1. SDS-PAGE analyses of in vitro translation products synthesized from UL1-specific RNA revealed that the UL1 ORF encodes a 30 kDa protein which corresponds in size to the 258 amino acid protein predicted by DNA sequence analyses. This result was confirmed by arresting translation of the in vitro transcribed UL1 RNA with an oligodeoxynucleotide complementary to UL1 coding sequences. The UL1 protein is a homolog of the predicted protein encoded by the ORF2 gene of varicella-zoster virus, but UL1 has no homolog in herpes simplex virus type 1. The UL1 protein contains a region conforming to a 'PEST' (Proline, Glutamic acid, Serine, and Threonine) sequence, which is commonly found in proteins with half-lives of less than two hours.

Identification and transcriptional analyses of the UL3 and UL4 genes of equine herpesvirus 1, homologs of the ICP27 and glycoprotein K genes of herpes simplex virus.

The DNA sequence of 3,240 nucleotides of the XbaI G fragment located in the unique long (UL) region of the equine herpesvirus 1 genome revealed two major open reading frames (ORFs) designated UL3 and UL4. The UL3 ORF of 470 amino acids (aa) maps at nucleotides (nt) 4450 to 3038 from the long terminus, and its predicted 51.4-kDa protein product exhibits significant homology to the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1; 32% identity) and to the ORF4 protein of varicella-zoster virus (13% identity). Interestingly, a zinc finger motif is conserved in the C-terminal domains of both ICP27 of HSV-1 (aa 483 to 508) and UL3 of equine herpesvirus 1 (aa 441 to 466). The UL4 ORF of 343 aa maps at nt 5618 to 4587 and could encode a protein of 38.1 kDa which exhibits significant homology to the UL53 protein (cell fusion protein or glycoprotein K) of HSV-1 (26% identity) and to the ORF5 protein of varicella-zoster virus (33% identity). Analyses of the UL4 amino acid sequence revealed domains characteristic of a membrane-bound glycoprotein and included potential signature sequences for (i) a signal sequence, (ii) two N-linked glycosylation sites, and (iii) four transmembrane domains. Nucleotide sequence analyses also revealed potential TATA boxes located upstream of the UL3 and UL4 ORFs. However, only a single polyadenylation signal (nt 2988 to 2983) was detected downstream of the UL3 ORF. Northern (RNA) blot hybridization and S1 nuclease analyses were used to map and characterize the UL3 and UL4 mRNAs. Metabolic inhibitors were used to identify the kinetic class of these two genes. The data revealed that UL3 is an early gene that encodes a 1.6-kb mRNA, while UL4 is a late gene encoding a 3.8-kb mRNA that overlaps the UL3 transcript. Both transcripts were shown by S1 nuclease analyses to initiate 24 to 26 nt downstream of their respective TATA boxes and to have a common transcription termination signal as a pair of 3'-coterminal mRNAs.

Identification and characterization of an equine herpesvirus 1 late gene encoding a potential zinc finger.

In this report, we present the DNA sequence and transcriptional characterization of a gene (IR5) that maps within each of the inverted repeat (IR) segments of the equine herpesvirus type 1 (EHV-1) genome. The IR5 open reading frame (ORF) is located within both IR sequences (nucleotides 9932-10,642 of the IR). DNA sequence analyses of the IR5 gene region revealed an ORF of 236 amino acids (24,793 Da) that showed significant homology to ORF64 of varicella-zoster virus and ORF3 of EHV-4 both of which map within the inverted repeats and to the US10 ORF of herpes simplex virus type 1 (HSV-1) which maps within the unique short segment. Additional analyses of the nucleotide sequence failed to reveal any overlapping ORFs that would correspond to US11 or US12 of HSV-1. Interestingly, the IR5 ORF of EHV-1 possesses a sequence of 13 amino acids (CAYWCCLGHAFAC) that is a perfect match to the consensus zinc finger motif (C-X2-4-C-X2-15-C/H-X2-4-C/H). Putative cis-acting elements flanking the IR5 ORF include a TATA box (nucleotides 9864-9870), a CAAT box (nucleotides 9709-9714), and a polyadenylation signal (nucleotides 10,645-10,650). Northern blot and S1 nuclease analyses identified a single 0.9-kb mRNA species that first appears at 2 hr postinfection, and whose synthesis is reduced in the presence of phosphonoacetic acid, an inhibitor of EHV-1 DNA synthesis. Thus, the IR5 gene of EHV-1 exhibits characteristics representative of a late gene of the gamma-1 class. The characterization of the IR5 gene at the DNA and RNA levels will facilitate ongoing studies to identify and characterize the IR5 polypeptide.

ICP22 homolog of equine herpesvirus 1: expression from early and late promoters.

The complete nucleotide sequence of the short region, made up of a unique segment (Us; 6.5 kb) bracketed by a pair of inverted repeat sequences (IR; 12.8 kb each), of the equine herpesvirus 1 (EHV-1) genome has been determined recently in our laboratory. Analysis of the IR segment revealed a major open reading frame (ORF) designated IR4. The IR4 ORF exhibits significant homology to the immediate-early gene US1 (ICP22) of herpes simplex virus type 1 and to the ICP22 homologs of varicella-zoster virus (ORF63), pseudorabies virus (RSp40), and equine herpesvirus 4 (ORF4). The IR4 ORF is located entirely within each of the inverted repeat sequences (nucleotides [nt] 7918 to 9327) and has the potential to encode a polypeptide of 469 amino acids (49,890 Da). Within the IR4 ORF are two reiterated sequences: a 7-nt sequence tandemly repeated 17 times and a 25-nt sequence tandemly repeated 13 times. Nucleotide sequence analyses of IR4 also revealed several potential cis-regulatory sequences, two TATA sequences separated by 287 nt, an in-frame translation initiation codon following each TATA sequence, and a single polyadenylation site. To address the nature of the mRNA species encoded by IR4, we used Northern (RNA) blot and S1 nuclease analyses. RNA mapping data revealed that IR4 has two promoters that are regulated differentially during a lytic infection. A 1.4-kb mRNA appears initially at 2 h postinfection and is an early transcript since its synthesis is not affected by the presence of phosphonoacetic acid, an inhibitor of EHV-1 DNA replication. In contrast, a 1.7-kb mRNA appears at later times postinfection and is designated as a gamma-1 transcript, since its synthesis is significantly reduced by phosphonoacetic acid. These IR4-specific mRNAs are 3' coterminal, have unique 5' termini, and would code for in-frame, overlapping, carboxy-coterminal proteins of 293 and 469 amino acids, respectively. Interestingly, the site of homologous recombination to generate the genome of EHV-1 defective interfering particles that initiate persistent infection occurs between nt 3244 and 3251 of UL3 (ICP27 homolog) and nt 9027 and 9034 of IR4 (ICP22 homolog). Thus, this recombination event would generate a unique ORF that would encode a potential protein whose amino end was derived from the N-terminal 193 amino acids of the ICP22 homolog and whose carboxyl end was derived from the C-terminal 68 amino acids of the ICP27 homolog.