Kaposi's sarcoma-associated herpesvirus ORF18 and ORF30 are essential for late gene expression during lytic replication.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. IMPORTANCE: While KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

The anti-interferon activity of conserved viral dUTPase ORF54 is essential for an effective MHV-68 infection.

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR-/- mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68.

Induction of protective immunity against murine gammaherpesvirus 68 infection in the absence of viral latency.

Human gammaherpesviruses, Epstein-Barr virus, and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus are important pathogens associated with diseases, including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) is used as an experimental model system to study the host immune control of infection and explore novel vaccine strategies based on latency-deficient live viruses. We studied the properties and the potential of a recombinant MHV-68 (AC-RTA) in which the genes required for persistent infection were replaced by a constitutively expressed viral transcription activator, RTA, which dictates the virus to lytic replication. After intranasal infection of mice, replication of AC-RTA in the lung was attenuated, and no AC-RTA virus or viral DNA was detected in the isolated splenocytes, indicating a lack of latency in the spleen. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. Importantly, vaccination of AC-RTA was able to protect mice against subsequent challenge by the wild-type MHV-68. AC-RTA provides a vaccine strategy for preventing infection of human gammaherpesviruses. Furthermore, our results suggest that immunity to the major latent antigens is not required for protection.

Construction and characterization of an infectious murine gammaherpesivrus-68 bacterial artificial chromosome.

Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.

ORF30 and ORF34 are essential for expression of late genes in murine gammaherpesvirus 68.

A hallmark of productive infection by DNA viruses is the coupling of viral late gene expression to genome replication. Here we report the identification of open reading frame 30 (ORF30) and ORF34 as viral trans factors crucial for activating late gene transcription following viral DNA replication during lytic infection of murine gammaherpesvirus 68 (MHV-68). The mutant virus lacking either ORF30 or ORF34 underwent normal DNA replication but failed to express viral late gene transcripts, leading to nonproductive infection. In a reporter assay system, ORF30 and ORF34 were required for MHV-68 to activate the viral late gene promoters. Furthermore, studies using chromatin immunoprecipitation assays showed that the recruitment of RNA polymerase II to the viral late promoters during lytic infection was significantly reduced in the absence of ORF30 or ORF34. Together, the results suggest that ORF30 and ORF34 may play an important role in the assembly of the transcription initiation complex at the late gene promoters. Our discovery of the viral mutants that uncouple late gene transcription from DNA replication lays an important foundation to dissect the mechanism of this critical step of gene expression regulation.

Deletion of immune evasion genes provides an effective vaccine design for tumor-associated herpesviruses.

Vaccines based on live attenuated viruses often induce broad, multifaceted immune responses. However, they also usually sacrifice immunogenicity for attenuation. It is particularly difficult to elicit an effective vaccine for herpesviruses due to an armament of immune evasion genes and a latent phase. Here, to overcome the limitation of attenuation, we developed a rational herpesvirus vaccine in which viral immune evasion genes were deleted to enhance immunogenicity while also attaining safety. To test this vaccine strategy, we utilized murine gammaherpesvirus-68 (MHV-68) as a proof-of-concept model for the cancer-associated human γ-herpesviruses, Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus. We engineered a recombinant MHV-68 virus by targeted inactivation of viral antagonists of type I interferon (IFN-I) pathway and deletion of the latency locus responsible for persistent infection. This recombinant virus is highly attenuated with no measurable capacity for replication, latency, or persistence in immunocompetent hosts. It stimulates robust innate immunity, differentiates virus-specific memory T cells, and elicits neutralizing antibodies. A single vaccination affords durable protection that blocks the establishment of latency following challenge with the wild type MHV-68 for at least six months post-vaccination. These results provide a framework for effective vaccination against cancer-associated herpesviruses through the elimination of latency and key immune evasion mechanisms from the pathogen.

Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo.

Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.

Conserved herpesviral kinase promotes viral persistence by inhibiting the IRF-3-mediated type I interferon response.

A conserved herpesviral kinase, designated ORF36 in murine gamma-herpesvirus 68 (MHV-68), plays multiple vital roles in the viral life cycle. Here, we show by screening mutant viruses that ORF36 counteracts the antiviral type I interferon (IFN) response. ORF36 specifically binds to the activated form of interferon regulatory factor 3 (IRF-3) in the nucleus, inhibiting IRF-3 interaction with the cotranscriptional activator CBP and thereby suppressing the recruitment of RNA polymerase II to the interferon beta promoter. The anti-IFN function of ORF36 is conserved among herpesvirus subfamilies, although the conserved kinase activity is not absolutely required for this function. MHV-68 lacking ORF36 induces a greater interferon response and is attenuated in vitro and in vivo, where acute viral infection in the lung and latency in the spleen are compromised. Our data suggest that herpesviruses have evolved within their conserved kinase an anti-IFN activity critical for evasion of host immunity and for persistence.

Generation of a latency-deficient gammaherpesvirus that is protective against secondary infection.

Kaposi's sarcoma-associated herpesvirus and murine gammaherpesvirus-68 (MHV-68) establish latent infections and are associated with various types of malignancies. They are members of the gamma-2 herpesvirus subfamily and encode a replication and transcriptional activator, RTA, which is necessary and sufficient to disrupt latency and initiate the viral lytic cycle in vitro. We have constructed a recombinant MHV-68 virus that overexpresses RTA. This virus has faster replication kinetics in vitro and in vivo, is deficient in establishing latency, exhibits a reduction in the development of a mononucleosis-like disease in mice, and can protect mice against challenge by wild-type MHV-68. The present study, by using MHV-68 as an in vivo model system, demonstrated that RTA plays a critical role in the control of viral latency and suggests that latency is a determinant of viral pathogenesis in vivo.

Transcription program of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68 [also referred to as gammaHV68]) is phylogenetically related to Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV). However, unlike KSHV or EBV, MHV-68 readily infects fibroblast and epithelial cell lines derived from several mammalian species, providing a system to study productive and latent infections as well as reactivation of gammaherpesviruses in vivo and in vitro. To carry out rapid genome-wide analysis of MHV-68 gene expression, we made DNA arrays containing nearly all of the known and predicted open reading frames (ORFs) of the virus. RNA obtained from an MHV-68 latently infected cell line, from cells lytically infected with MHV-68 in culture, and from the lung tissue of infected mice was used to probe the MHV-68 arrays. Using a tightly latent B-cell line (S11E), the MHV-68 latent transcription program was quantitatively described. Using BHK-21 cells and infected mice, we demonstrated that latent genes are transcribed during lytic replication and are relatively independent of de novo protein synthesis. We determined that the transcription profiles at the peak of lytic gene expression are similar in cultured fibroblast and in the lung of infected mice. Finally, the MHV-68 DNA arrays were used to examine the gene expression profile of a recombinant virus that overexpresses replication and transcription activator (RTA), C-RTA/MHV-68, during lytic replication in cell culture. The recombinant virus replicates faster then the parental strain and the DNA arrays revealed that nearly every MHV-68 ORF examined was activated by RTA overexpression. Examination of the gene expression patterns of C-RTA/MHV-68 over a time course led to the finding that the M3 promoter is RTA responsive in the absence of other viral factors.