Interferon Gamma Inhibits Equine Herpesvirus 1 Replication in a Cell Line-Dependent Manner.

The sole equine herpesvirus 1 (EHV-1) immediate-early protein (IEP) is essential for viral replication by transactivating viral immediate-early (IE), early (E), and late (L) genes. Here, we report that treatment of mouse MH-S, equine NBL6, and human MRC-5 cells with 20 ng/mL of IFN-γ reduced EHV-1 yield by 1122-, 631-, and 10,000-fold, respectively. However, IFN-γ reduced virus yield by only 2-4-fold in mouse MLE12, mouse L-M, and human MeWo cells compared to those of untreated cells. In luciferase assays with the promoter of the EHV-1 early regulatory EICP0 gene, IFN-γ abrogated trans-activation activity of the IEP by 96% in MH-S cells, but only by 21% in L-M cells. Similar results were obtained in assays with the early regulatory UL5 and IR4 promoter reporter plasmids. IFN-γ treatment reduced IEP protein expression by greater than 99% in MH-S cells, but only by 43% in L-M cells. The expression of IEP and UL5P suppressed by IFN-γ was restored by JAK inhibitor treatment, indicating that the inhibition of EHV-1 replication is mediated by JAK/STAT1 signaling. These results suggest that IFN-γ blocks EHV-1 replication by inhibiting the production of the IEP in a cell line-dependent manner. Affymetrix microarray analyses of IFN-γ-treated MH-S and L-M cells revealed that five antiviral ISGs (MX1, SAMHD1, IFIT2, NAMPT, TREX1, and DDX60) were upregulated 3.2-18.1-fold only in MH-S cells.

Intranasal treatment with CpG-B oligodeoxynucleotides protects CBA mice from lethal equine herpesvirus 1 challenge by an innate immune response.

Equine herpesvirus 1 (EHV-1) is the causative agent of a number of equine disease manifestations, including severe disease of the central nervous system, respiratory infections, and abortion storms. Our results showed that intranasal treatment with CpG-B oligodeoxynucleotides (ODN 1826) protected CBA mice from pathogenic EHV-1 RacL11 challenge. The IFN-γ gene and seven interferon-stimulated genes (ISGs) were upregulated 39.4- to 260.3-fold at 8 h postchallenge in the lungs of RacL11-challenged mice that had been treated with CpG-B ODN. Interestingly, IFN-γ gene expression was upregulated by 26-fold upon RacL11 challenge in CpG-B ODN-treated mice lungs as compared to that of CpG-A ODN (ODN 1585)-treated mice lungs; however, the seven ISGs were upregulated by 2.4-5.0-fold, suggesting that IFN-γ is a major factor in the protection of CBA mice from the lethal challenge. Pre-treatment with IFN-γ significantly reduced EHV-1 yield in murine alveolar macrophage MH-S cells, but not in mouse lung epithelial MLE12 cells. These results suggest that CpG-B ODN may be used as a prophylactic agent in horses and provide a basis for more effective treatment of EHV-1 infection.

Interferon Gamma Inhibits Varicella-Zoster Virus Replication in a Cell Line-Dependent Manner.

The major immediate early 62 (IE62) protein of varicella-zoster virus (VZV) is delivered to newly infected cell nuclei, where it initiates VZV replication by transactivating viral immediate early (IE), early (E), and late (L) genes. Interferon gamma (IFN-γ) is a potent cytokine produced following primary VZV infection. Furthermore, VZV reactivation correlates with a decline in IFN-γ-producing immune cells. Our results showed that treatment with 20 ng/ml of IFN-γ completely reduced intracellular VZV yield in A549 lung epithelial cells, MRC-5 lung fibroblasts, and ARPE-19 retinal epithelial cells at 4 days post-VZV infection. However, IFN-γ reduced virus yield only 2-fold in MeWo melanoma cells compared to that of untreated cells. IFN-ß significantly inhibited VZV replication in both ARPE-19 and MeWo cells. In luciferase assays with VZV open reading frame 61 (ORF61) promoter reporter plasmid, IFN-γ abrogated the transactivation activity of IE62 by 95%, 97%, and 89% in A549, ARPE-19, and MRC-5 cells, respectively. However, IFN-γ abrogated IE62's transactivation activity by 16% in MeWo cells, indicating that IFN-γ inhibits VZV replication as well as IE62-mediated transactivation in a cell line-dependent manner. The expression of VZV IE62 and ORF63 suppressed by IFN-γ was restored by JAK1 inhibitor treatment, indicating that the inhibition of VZV replication is mediated by JAK/STAT1 signaling. In the presence of IFN-γ, knockdown of interferon response factor 1 (IRF1) increased VZV replication. Ectopic expression of IRF1 reduced VZV yields 4,000-fold in MRC-5 and ARPE-19 cells but 3-fold in MeWo cells. These results suggest that IFN-γ blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner.IMPORTANCE Our results showed that IFN-γ significantly inhibited VZV replication in a cell line-dependent manner. IFN-γ inhibited VZV gene expression after the immediate early stage of infection and abrogated IE62-mediated transactivation. These results suggest that IFN-γ blocks VZV replication by inhibiting IE62 function in a cell line-dependent manner. Understanding the mechanisms by which IFN-γ plays a role in VZV gene programming may be important in determining the tissue restriction of VZV.

Comparative Genomic Sequencing and Pathogenic Properties of Equine Herpesvirus 1 KyA and RacL11.

Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide. The virus causes respiratory disease, abortion, and, in some cases, neurological disease. EHV-1 Kentucky A (KyA) is attenuated in the mouse and equine, whereas wild-type pathogenic strain RacL11 induces severe inflammatory infiltration of the lung, causing infected mice to succumb. The complete DNA sequencing of the KyA genome revealed that genes UL17 (ORF17), US6 (ORF73; gI), US7 (ORF74; gE), and US8 (ORF75; 10 K) are deleted as compared to the RacL11 and Ab4 genomes. In-frame deletions in the US1 (ORF68), US4 (ORF71; gp2), and UL63 (ORF63; EICP0) genes and point mutations in 14 different open reading frames (ORFs) were detected in the KyA genome. Interestingly, UL1 (ORF1) and UL2 (ORF2) were deleted in both KyA and RacL11. Our previous studies showed that EHV-1 glycoproteins gI, gE, and full-length gp2 contribute to the pathogenesis of the RacL11 strain. The confirmation of these gene deletions in KyA suggests their contribution to the attenuation of this virus. The growth kinetics results revealed that KyA replicates to high titers in cell culture as compared to RacL11 and Ab4, indicating that the above genomic deletions and mutations in KyA do not have an inhibitory effect on KyA replication in cells of mouse, rabbit, equine, or human origin. Studies of EHV-1 pathogenesis in CBA mice showed that KyA is attenuated whereas mice infected with RacL11 succumbed by 3-6 days post-infection, which is consistent with our previous results.

Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses That Protect Mice from Lethal Challenge.

UNLABELLED: Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide. The virus causes respiratory disease, abortion, and, in some cases, neurological disease. EHV-1 strain KyA is attenuated in the mouse and equine, whereas wild-type strain RacL11 induces severe inflammation of the lung, causing infected mice to succumb at 4 to 6 days postinfection. Our previous results showed that KyA immunization protected CBA mice from pathogenic RacL11 challenge at 2 and 4 weeks postimmunization and that KyA infection elicited protective humoral and cell-mediated immune responses. To investigate the protective mechanisms of innate immune responses to KyA, KyA-immunized mice were challenged with RacL11 at various times postvaccination. KyA immunization protected mice from RacL11 challenge at 1 to 7 days postimmunization. Immunized mice lost less than 10% of their body weight and rapidly regained weight. Virus titers in the lungs of KyA-immunized mice were 1,000-fold lower at 2 days post-RacL11 challenge than virus titers in the lungs of nonimmunized mice, indicating accelerated virus clearance. Affymetrix microarray analysis revealed that gamma interferon (IFN-γ) and 16 antiviral interferon-stimulated genes (ISGs) were upregulated 3.1- to 48.2-fold at 8 h postchallenge in the lungs of RacL11-challenged mice that had been immunized with KyA. Murine IFN-γ inhibited EHV-1 infection of murine alveolar macrophages and protected mice against lethal EHV-1 challenge, suggesting that IFN-γ expression is important in mediating the protection elicited by KyA immunization. These results suggest that EHV-1 KyA may be used as a live attenuated EHV-1 vaccine as well as a prophylactic agent in horses. IMPORTANCE: Viral infection of cells initiates a signal cascade of events that ultimately attempts to limit viral replication and prevent infection through the expression of host antiviral proteins. In this study, we show that EHV-1 KyA immunization effectively protected CBA mice from pathogenic RacL11 challenge at 1 to 7 days postvaccination and increased the expression of IFN-γ and 16 antiviral interferon-stimulated genes (ISGs). The administration of IFN-γ blocked EHV-1 replication in murine alveolar macrophages and mouse lungs and protected mice from lethal challenge. To our knowledge, this is the first report of an attenuated EHV-1 vaccine that protects the animal at 1 to 7 days postimmunization by innate immune responses. Our findings suggested that IFN-γ serves as a novel prophylactic agent and may offer new strategies for the development of anti-EHV-1 agents in the equine.

Full trans-activation mediated by the immediate-early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence.

The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt -89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS).

Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62.

UNLABELLED: The immediate early 62 protein (IE62) of varicella-zoster virus (VZV), a major viral trans-activator, initiates the virus life cycle and is a key component of pathogenesis. The IE62 possesses several domains essential for trans-activation, including an acidic trans-activation domain (TAD), a serine-rich tract (SRT), and binding domains for USF, TFIIB, and TATA box binding protein (TBP). Transient-transfection assays showed that the VZV IE62 lacking the SRT trans-activated the early VZV ORF61 promoter at only 16% of the level of the full-length IE62. When the SRT of IE62 was replaced with the SRT of equine herpesvirus 1 (EHV-1) IEP, its trans-activation activity was completely restored. Herpes simplex virus 1 (HSV-1) ICP4 that lacks a TAD very weakly (1.5-fold) trans-activated the ORF61 promoter. An IE62 TAD-ICP4 chimeric protein exhibited trans-activation ability (10.2-fold), indicating that the IE62 TAD functions with the SRT of HSV-1 ICP4 to trans-activate viral promoters. When the serine and acidic residues of the SRT were replaced with Ala, Leu, and Gly, trans-activation activities of the modified IE62 proteins IE62-SRTΔSe and IE62-SRTΔAc were reduced to 46% and 29% of wild-type activity, respectively. Bimolecular complementation assays showed that the TAD of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with Mediator 25 in human melanoma MeWo cells. The SRT of IE62 interacted with the nucleolar-ribosomal protein EAP, which resulted in the formation of globular structures within the nucleus. These results suggest that the SRT plays an important role in VZV viral gene expression and replication. IMPORTANCE: The immediate early 62 protein (IE62) of varicella-zoster virus (VZV) is a major viral trans-activator and is essential for viral growth. Our data show that the serine-rich tract (SRT) of VZV IE62, which is well conserved within the alphaherpesviruses, is needed for trans-activation mediated by the acidic trans-activation domain (TAD). The TADs of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with cellular Mediator 25 in bimolecular complementation assays. The interaction of the IE62 SRT with nucleolar-ribosomal protein EAP resulted in the formation of globular structures within the nucleus. Understanding the mechanisms by which the TAD and SRT of IE62 contribute to the function of this essential regulatory protein is important in understanding the gene program of this human pathogen.

The EHV-1 UL4 protein that tempers viral gene expression interacts with cellular transcription factors.

The UL4 gene is conserved within the genome of defective interfering particles of equine herpesvirus type 1 (EHV-1) that mediate persistent infection. Here, we show that the UL4 protein inhibits EHV-1 reporter gene expression by decreasing the level of transcribed mRNA. The UL4 protein did not bind any gene class of EHV-1 promoters in electromobility or chromatin immunoprecipitation assays, but directly interacted with the TATA box-binding protein (TBP) and the carboxy-terminal domain of RNA polymerase II both in vitro (GST-pulldown assays) and in infected cells (coimmunoprecipitation analyses). Microarray analyses of the expression of the 78 EHV-1 genes revealed that viral late genes important for virion assembly displayed enhanced expression in cells infected with UL4-null virus as compared to wild-type or UL4-restored EHV-1. Quantitative PCR analyses showed that viral DNA replication was not retarded in cells infected with the UL4-null virus as compared to wild-type EHV-1.

Deletion of the UL4 gene sequence of equine herpesvirus 1 precludes the generation of defective interfering particles.

Serial, high multiplicity passage of equine herpesvirus 1 (EHV-1) leads to the generation of defective interfering particles (DIP). EHV-1 DIP inhibit and interfere with the replication of standard EHV-1, establishing a state of persistent infection. These DIP package severely truncated and rearranged forms of the standard viral genome. Contained within the DIP genome are only three genes: UL3, UL4, and a unique hybrid gene (Hyb). The hybrid gene forms through a recombination event that fuses portions of the early regulatory IR4 and UL5 genes and is essential for DIP-mediated interference. The UL4 gene is an early gene dispensable for lytic replication and inhibits viral and cellular gene expression. However, the contribution of the UL4 gene during DIP-mediated persistent infection is unknown. Here, we describe the generation of a completely deleted UL4 virus and its use to investigate the role of the UL4 gene in the generation of the defective genome. Deletion of the UL4 gene resulted in delayed virus growth at late times post-infection. Cells infected with a mutant EHV-1 that lacked expression of the UL4 protein due to an inserted stop codon in the UL4 gene produced defective particles, while cells infected with a mutant EHV-1 that had the complete UL4 gene sequence deleted were unable to produce DIP. These data suggest that the UL4 gene sequence, but not the UL4 protein, is critical for the generation of defective interfering particles.

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus.

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

Development of a bacterial artificial chromosome (BAC) recombineering procedure using galK-untranslated region (UTR) for the mutation of diploid genes.

Bacterial artificial chromosome (BAC) recombineering using galK selection allows DNA cloned in Escherichia coli to be modified without introducing an unwanted selectable marker at the modification site. Genomes of some herpesviruses have a pair of inverted repeat sequences that makes it very difficult to introduce mutations into diploid (duplicate) genes using the galK selection method. To mutate diploid genes, we developed a galK-UTR BAC recombineering procedure that blocks one copy of the target diploid gene by insertion of a galK untranslated region (UTR), which enables the simple mutation of the other copy. The blocked copy can then be replaced with an UTR-specific primer pair. The IR2 gene of equine herpesvirus 1 (EHV-1) maps within both the internal (IR) and terminal repeat (TR) of the genomic short region and is expressed at low levels because its promoter is TATA-less. Both IR2 promoters in EHV-1 BAC were replaced with a mutant IR2 promoter containing three Sp1-binding motifs and a consensus TATA box by galK-UTR BAC recombineering. The expression level of the IR2 protein controlled by the modified promoter increased approximately 4-fold as compared to that of wild-type EHV-1. The galK-UTR method will provide a useful tool in studies of herpesviruses.

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene.

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5'-ATCGT-3') located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box.

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 ~33kDa 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.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication.

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain.

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 4hours 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.

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.

The equine herpesvirus-1 (EHV-1) IR3 transcript downregulates expression of the IE gene and the absence of IR3 gene expression alters EHV-1 biological properties and virulence.

The IR3 transcript of equine herpesvirus-1 (EHV-1) harbors 117 nts antisense to the immediate-early (IE) mRNA, suggesting it plays a regulatory role. Here, we show that the IR3 transcript downregulates IE gene expression and that the absence of IR3 expression altered EHV-1 biological properties and virulence in mice. Reporter assays revealed that the IR3/IE overlapping sequences [IR3(+226/+342)] and an additional IR3(+343/+433) region are necessary for the IR3 RNA to downregulate IE expression. Experiments with the DeltaIR3 EHV-1 showed that the IR3 gene is dispensable for EHV-1 replication. Protein expression of the IE and representative EHV-1 genes was increased in cells infected with DeltaIR3 EHV-1 as compared to that of cells infected with wt EHV-1. The DeltaIR3 EHV-1 exhibited increased virulence in mice as compared to the parent virus. The finding that the IR3 transcript affects IE gene expression extends the role of RNA as a regulatory molecule in alphaherpesvirus infection.

The IR4 auxiliary regulatory protein expands the in vitro host range of equine herpesvirus 1 and is essential for pathogenesis in the murine model.

IR4, an early regulatory protein of equine herpesvirus 1 (EHV-1), is not a DNA-binding protein, but interacts with the sole immediate-early protein (IEP) to increase both IEP site-specific DNA-binding and IEP-mediated trans-activation of EHV-1 promoters. To investigate the biological properties of IR4 and ascertain whether this regulatory protein is essential for virus growth, bacterial artificial chromosome methods were employed to generate an IR4-null EHV-1. The IR4 gene was dispensable for EHV-1 growth in non-immortalized equine NBL-6 cells, but virus replication was delayed and was reduced by greater than 10-fold. In addition, replication of the IR4 mutant was abrogated in all other cell types tested, including equine ETCC tumor cells and cells of mouse, rabbit, monkey, and human origin. Further, in contrast to the highly pathogenic parent virus, the IR4 deletion mutant failed to cause disease in the CBA mouse as judged by assessing body weight and clinical signs and was unable to replicate in the murine lung. To define the nature of the block in the replication of the IR4-null virus, molecular analyses were carried out in RK-13 rabbits' cells infected with the IR4-deleted virus and revealed that: 1) the synthesis of the sole IEP was not inhibited; 2) the synthesis of early viral proteins examined was either not affected or was delayed to late times; 3) viral DNA replication was inhibited by more than 99.9%; and 4) synthesis of essential late proteins such as glycoprotein D and glycoprotein K was prevented. These findings indicate that the IR4 protein is required for EHV-1 DNA replication in non-permissive cells, and, like its homologues in other alphaherpesviruses, contributes a function required for virus replication in a variety of cell types.

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.