The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters

Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 (EICP22) and UL5 (EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4 hours post-infection, was localized throughout the nucleus and cytoplasm, and was not present in purified virions. EHV-1 lacking UL4P expression was infectious and displayed cell tropism and pathogenic properties in the mouse model similar to those of parental and revertant viruses. Reporter assays demonstrated that the UL4P has a broad inhibitory function, suggesting a potential role in establishing and/or maintaining DIP-mediated persistent infection.     

Biological and genotypic properties of defective interfering particles of equine herpesvirus 1 that mediate persistent infection

Infection with equine herpesvirus 1 (EHV-1) preparations enriched for defective interfering particles (DIP) leads to a state of persistent infection in which infected cells become lysis resistant and release both infectious (standard) virus and DIP. EHV-1 DIP are unique in that the recombination events that generate DIP genomes produce new open reading frames (ORFs; Hyb1.0 and Hyb2.0) consisting of 5′ sequences of varying lengths of the early regulatory gene IR4 fused to 3′ sequences of varying lengths of the UL5 regulatory gene. Only two additional ORFs (UL3 and UL4) are conserved. Because persistently infected cells release a heterogeneous mixture of DIP, characterization of the elements responsible for this altered state of infection has proved difficult. Here we describe a method for studying persistent infection using recombinant DIP (rDIP). Infection with rDIP resulted in the production of recombinant DIP that replicated faithfully to, at least, five passages and mediated a rapid progression to persistent infection as measured by: 1) production of cells resistant to lysis by the standard virus; and 2) infected cells that released both standard virus and DIP. High concentrations of rDIP also resulted in interference with the standard virus replication, another hallmark of persistent infection. rDIP deleted of UL3, UL4, and either Hyb gene, the only functional genes conserved in the DIP genome, replicated but exhibited markedly reduced ability to interfere with standard virus replication. Restoring only the Hyb genes (either Hyb1.0 or Hyb2.0), the IR4 gene, or specific portions of the IR4 gene restored interference. These data suggest that residues 144 to 196 of the IR4 protein within the HYB proteins are important for DIP interference and that persistent infection results from recombination events that produce DIP genomes.     

Organization and function of the ORIs sequence in the genome of EHV-1 DI particles

Equine herpesvirus type 1 (EHV-1) cultures enriched for defective interfering particles (DIP) mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. We have recently demonstrated that an origin of replication (ORI) is located within the central portion (map units 0.828 and 0.948) of the inverted repeat sequence (IRs) of the short region of the standard EHV-1 genome. In the generation of the genome of EHV-1 DI particles, sequences from this internal portion of the IRs recombine with sequences at the long region terminus at nucleotides 3244-3251. In this paper we report that the ORIs sequence is precisely conserved in the DIP genome, that direct repeat sequences near the ORIs sequence which may enhance DNA replication are mutated in the DIP genome, and that the ORI sequence of DIP DNA is functional in DNA replication assays.     

Genetic Complexity of EHV-1 Defective Interfering Particles and Identification of Novel IR4/UL5 Hybrid Proteins Produced During Persistent Infection

This study examined the genetic complexity of three equine herpesvirus 1 (EHV-1) defective interfering particles (DIP) and found the DIP genomes to range from 5.9 kbp to 7.3 kbp in total size. Each DIP contains an identical 5′ end (∼1.9 kb) that harbors UL3 and UL4 genes that are 100% identical to those of the infectious virus. DIP2 and DIP3 contain a previously described unique IR4/UL5 (EICP22/EICP27) hybrid gene (Hyb1.0). The DIP1 genome, however, appears to be generated from a different recombination event which results in the formation of a new distinct hybrid ORF. The new ORF (Hyb2.0) is comprised of 684 bp from the 5′ end of IR4 fused to 45 bp from the 3′ terminus of UL5. In contrast to Hyb1.0, the UL5 sequences present in Hyb2.0 are not in-frame. Thus, the Hyb2.0 protein is comprised of 228 residues from IR4 linked to a sequence of 15 amino acids that result from a frameshifted reading of UL5 sequences. Western blot analysis confirmed that the Hyb2.0 ORF is expressed during persistent infection to produce a family of proteins that migrate at 36–42 kDa. Fluorescence microscopy revealed that both Hyb proteins display diffuse cytoplasmic localization patterns dissimilar to the nuclear localization patterns of both IR4 and UL5. Neither Hyb protein, however, disrupts the nuclear entry of the EHV-1 immediate-early, IR4, or UL5 proteins or cellular TATA box binding protein (TBP) previously shown to interact with both IR4 or UL5 in productive infection. DIP genomic segments (∼3.5–5.0 kbp) downstream of the 100% conserved origin of replication are highly variable among the three DIP genomes and contain large areas of repetitive sequences. The possibility that the non-coding sequences play a role in viral interference and/or persistent infection remains to be determined.     

The early UL3 gene of equine herpesvirus-1 encodes a tegument protein not essential for replication or virulence in the mouse

The UL3 gene of equine herpesvirus-1 (EHV-1) is retained in the genome of defective interfering particles and encodes a ~ 33 kDa myristylated protein. Further characterization showed that the UL3 gene is trans-activated only by the sole immediate early (IE) protein and encodes an early protein that is dispensable for EHV-1 replication and localizes in the tegument of purified virions. UL3-deleted EHV-1 (vL11ΔUL3) exhibits properties of host cell tropism, plaque size, and growth kinetics similar to those of the parental virus. Expression levels of EHV-1 proteins representative of all three gene classes in vL11ΔUL3-infected cells were identical to those in cells infected with parental virus. Mice intranasally infected with vL11ΔUL3 and parental virus showed no significant difference in mortality or virus lung titers. These findings suggest that the UL3 protein does not play a major role in the biology of EHV-1 in cell culture or virulence in the mouse.     

The defective interfering particles of equine herpesvirus 1 encode an ICP22/ICP27 hybrid protein that alters viral gene regulation

The genomes of equine herpesvirus 1 (EHV-1) defective interfering (DI) particles that mediate persistent infection were shown to encode a unique hybrid open reading frame composed of sequences that encode the 196 N-terminal amino acids of ICP22 linked in-frame to the C-terminal 68 amino acids of ICP27. Previous studies demonstrated that this hybrid gene, designated as ICP22/ICP27. was expressed abundantly at both the mRNA and the protein levels in DI particle-enriched infections, but not in standard EHV-1 infection (Chen et al., 1996 J. Virol. 70, 313-320). Since the ICP22/ICP27 hybrid protein contains portions of two EHV-1 early regulatory proteins, its effect on EHV-1 gene regulation was investigated. In EHV-1-infected cells, the ICP22/ICP27 hybrid protein expressed from plasmid vectors significantly reduced expression of a reporter gene under the control of the EHV-1 immediate-early (IE) gene promoter and early gene promoter, such as the viral ICP27 gene. In uninfected cells, the ICP22/ICP27 hybrid protein moderately down-regulated the IE and ICP22 promoters, up-regulated late gene promoters such as IR5, and altered the regulatory function of the IE and 1CP22 proteins in co-transfected cells. These results demonstrated that DI particles might alter viral gene regulation by expression of a unique hybrid gene encoded on the DI particle genome.     

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase

Summary. The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy. Received September 2, 1997 Accepted January 14, 1998     

Challenging successive mosquito generations with a densonucleosis virus yields progressive survival improvement but persistent, innocuous infections

Research on cultivated shrimp suggests that they have the capability to tolerate viral pathogens in a highly specific manner by mechanisms currently unknown. The phenomenon is difficult to study in detail because they have a generation time of 1-2yr and lack continuous cell lines. Thus, we developed a mosquito-densovirus model to examine whether similar phenomena occur in insects. Serial challenge of five generations with a stock densovirus (AThDNV) resulted in progressive survival increases from 15% to 58%. Prevalence of AThDNV infection in surviving mosquito larvae (confirmed by PCR, histology, in situ hybridization and transmission electron microscopy) was relatively high (e.g. 36% in F2) but they grew normally to establish each succeeding generation. At the end of five generations, comparison of deduced amino acid sequences from genome fragments revealed a significantly higher (p=0.02) estimated prevalence of defective targets in the survivor virus population (29.7%+/-10.0 SD) than in the stored viral population (3.3%+/-5.8 SD). The results paralleled those reported for serially passaged C6/36 mosquito cell cultures infected with a densovirus. There, reduced infection rates are ascribed to the production of defective interfering particles (DIP). Thus, it is possible that the presence of prior AThDNV infections with a high level of DIP contributed to improved survival in our challenged F4 mosquito population. If so, it suggests that persistent viral infections in arthropods may serve in a specific, adaptive manner to reduce the incidence and severity of disease.     

Defective viral RNAs in Aedes albopictus C6/36 cells persistently infected with Semliki Forest virus

A persistent infection of Semliki Forest virus (SFV) has been established in Aedes albopictus C6/36 cells. Only a small number of cells survived the initial infection with this RNA virus and gave rise to a persistently infected culture which produced continuously small amounts of infectious virus. To investigate whether defective viral RNA is involved in establishing and maintaining a persistent infection, the intracellular viral RNA was analyzed early and late after infection by blot hybridizations. Several defective viral RNAs were detected with a common sequence corresponding to the 3′ end of the viral genome during and after the establishment of the persistent infection. These defective viral RNAs resemble the defective interfering RNAs in vertebrate cells generated during serial undiluted passages of standard SFV. The defective viral RNAs are rarely released from cells as virions. The rapid generation of defective viral RNAs may be important for the establishment of a persistent infection in mosquito cells.     

Generation and characterization of an EICP0 null mutant of equine herpesvirus 1

The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not essential for KyA replication in cell culture. Experiments to assess the effect of the EICP0 deletion on EHV-1 gene programming revealed that mRNA expression of the immediate-early gene and representative early and late genes as well as the synthesis of these viral proteins were reduced as compared to the kinetics of viral mRNA and protein synthesis observed for the wild type virus. However, the transition from early to late viral gene expression was not prevented or delayed, suggesting that the absence of the EICP0 gene did not disrupt the temporal aspects of EHV-1 gene regulation. The extracellular virus titer and plaque areas of the EICP0 mutant virus KyADeltaEICP0, in which the gp2-encoding gene 71 gene that is absent in the KyA BAC was restored, were reduced by 10-fold and 19%, respectively, when compared to parental KyA virus; while the titer and plaque areas of mutant KyADeltaEICP0Deltagp2 that lacks both the EICP0 gene and gene 71 were reduced more than 50-fold and 67%, respectively. The above results show that the EICP0 gene is dispensable for EHV-1 replication in cell culture, and that the switch from early to late viral gene expression for the representative genes examined does not require the EICP0 protein, but that the EICP0 protein may be structurally required for virus egress and cell-to-cell spread.     

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

The 150 kbp genome of equine herpesvirus-1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7 kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus.     

Defective interfering viruses and their potential as antiviral agents

Defective interfering (DI) virus is simply defined as a spontaneously generated virus mutant from which a critical portion of the virus genome has been deleted. At least one essential gene of the virus is deleted, either in its entirety, or sufficiently to make it non‐functional. The resulting DI genome is then defective for replication in the absence of the product(s) of the deleted gene(s), and its replication requires the presence of the complete functional virus genome to provide the missing functions. In addition to being defective DI virus suppresses production of the helper virus in co‐infected cells, and this process of interference can readily be observed in cultured cells. In some cases, DI virus has been observed to attenuate disease in virus‐infected animals. In this article, we review the properties of DI virus, potential mechanisms of interference and progress in using DI virus (in particular that derived from influenza A virus) as a novel type of antiviral agent. Copyright © 2009 John Wiley & Sons, Ltd.     

The equine herpesvirus 1 UL45 homolog encodes a glycosylated type II transmembrane protein and is involved in virus egress

Experiments to analyze the product of the equine herpesvirus type 1 (EHV-1) UL45 homolog were conducted. Using an antiserum generated against the carboxylterminal 114 amino acids of the EHV-1 UL45 protein, proteins of M(r) 32,000, 40,000, and 43,000 were detected specifically in EHV-1-infected cells. Neither form of the protein was located in purified virions of EHV-1 wild-type strain RacL22 or the modified live vaccine strain RacH, but UL45 was demonstrated to be expressed as a late (gamma-2) protein. Fractionation of infected cells and deglycosylation experiments demonstrated that the EHV-1 UL45 protein represents a type II membrane glycoprotein. Deletion of the UL45 gene in RacL22 and RacH (LDelta45 and HDelta45) showed that UL45 is nonessential for EHV-1 growth in vitro, but that deletion reduced the viruses' replication efficiency. A marked reduction of virus release was observed although no significant influence was noticed either on plaque size or on the syncytial phenotype of the EHV-1 strain RacH.     

Identification of the site of recombination in the generation of the genome of DI particles of equine herpesvirus type 1

Defective interfering particles (DIPs) are generated by serial, undiluted propagation of equine herpesvirus type 1 (EHV-1). DIP-rich preparations of EHV-1 mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. The defective genomes consist of reiterations of sequences from the left terminus (0.00 to 0.04 map units) of the long (L) region covalently linked to sequences from the inverted repeats (0.78 to 0.79, 0.83 to 0.87, 0.91 to 0.95, and 0.99 to 1.00 map units) of the short (S) region of the standard genome. We have identified and determined the nucleotide sequences of these segments of the standard genome as well as the component of the defective DNA that contains the site at which these two viral sequences recombined. Comparison of these sequences revealed that there is an 8-nucleotide sequence that is common to both the left terminus sequences and the inverted repeat sequences. These 8-nucleotide identical sequences are located at 3.25 kbp from the left terminus and at 9 kbp downstream of the L-S junction. The recombination between the left terminus and the inverted repeat sequences occurred at the site of homology and resulted in the generation of a novel open reading frame. The last 97 amino acids of an open reading frame of 469 amino acids encoded by sequences within the inverted repeats were replaced by a sequence of 68 amino acids encoded by a 204-bp sequence mapping at 0.023 map units. It will be of interest to determine whether this altered open reading frame, generated by recombination of sequences separated by more than 110,000 bp in the standard genome, plays a role in the varied outcomes of infection mediated by EHV-1 DIPs.     

Characterisation of IE and UL5 gene products of equine herpesvirus 1 using DNA inoculation of mice

Summary.  The equine herpesvirus 1 (EHV-1) strain HVS25A regulatory genes IE and UL5, encoding homologues of herpes simplex virus 1 (HSV-1) ICP4 and ICP27 respectively, were cloned into a eukaryotic expression vector and the DNA injected intramuscularly into mice. Antibodies produced in this way detected the IE or UL5 gene products as diffuse material in nuclei of RK13 cells transfected with the individual genes but as discrete punctate or large aggregates in RK13 cells infected with EHV-1. Western blotting on EHV-1 infected RK13 cells showed multiple IE products of 120–200 kDa and a UL5 product of 52 kDa. Inoculation with plasmids expressing EHV-1 IE or UL5 provided limited protection against EHV-1 challenge in mice as determined by increased virus clearance from lungs on day 2 post-challenge and a reduction in severity of lung histopathology. However, this protection was relatively weak compared with that provided by inoculation of DNA encoding EHV-1 glycoprotein D (gD), possibly reflecting the importance of neutralising antibody in this model. Accepted May 19, 2000 Received March 20, 2000     

Disruption of the gene encoding the gammaHV68 v-GPCR leads to decreased efficiency of reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68; MHV68) infection of mice has been a useful model for characterizing the role of conserved herpesvirus genes in pathogenesis. One of the well conserved genes among gamma2-herpesvirus, gene 74, encodes a viral G-protein coupled receptor (v-GPCR). To examine the role of the gammaHV68 v-GPCR in pathogenesis we have generated a mutant virus in which 440 base pairs of the gene 74 open reading frame have been deleted (gammaHV68v-GPCRDelta440). This deletion did not affect the growth of the virus in single or multiple rounds of replication in vitro, nor acute replication in vivo as assessed by plaque assay of spleens and lungs on days 4, 7 and 9 post-infection (p.i.). The ability of the v-GPCR mutant virus to establish latency and to reactivate from latency was quantitated on days 16 and 42 p.i. While there was no detectable difference in the ability of the mutant virus to either establish latency or reactivate from latency on day 16 p.i., as compared to wild-type gammaHV68 and marker rescue virus, there was a significant decrease in the efficiency of virus reactivation by day 42 p.i. Notably, mice infected with the mutant virus lacking the v-GPCR contained a higher frequency of viral genome positive cells in the peritoneum by day 42 p.i. than mice infected with either wild type or marker rescue virus. However, analysis of virus reactivation demonstrated that approximately the same frequency of cells reactivated virus from mice infected with either the gammaHV68 v-GPCR mutant, wild-type virus, or marker rescue virus. From these experiments we conclude that the gammaHV68 v-GPCR is dispensable for acute virus replication in vivo, but does play a role in reactivation from latency.     

Defective interfering particles: effects in modulating virus growth and persistence

Defective interfering virus particles (DIP) frequently play an important part in viral persistence in vitro, and may in some instances modify a virus infection in vivo, causing attenuation or persistence of the infection. To explain certain aspects of the growth of these mutants in vitro, other factors have been invoked such as interferon, mutations in the wild-type virus or the infected cells, or other substances released by infected cells that attenuate the infection. We present here a simple model of the growth of DIP in vitro which shows that (a) the observed population dynamics of DIP can readily be explained without invoking such extrinsic factors; (b) the initial multiplicity of infection of DIP is the principal determinant of the outcome of infection in both single- and repeated-passage cultures; and (c) in a long-term culture in vitro, the criterion used to decide the time of virus passage directly determines how long the standard virus, DIP, and cells survive. This model may be used with minor modifications to predict the behavior in vitro of other mutant viruses with a dominantly interfering phenotype.