Interactions Between Cancer-associated Gammaherpesvirus And Host:Lessons From DIP Virus

Background and objectives:Human gammaherpesviruses including Epstein-Barr virus (EBV/HHV-4) and Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) are closely related to a number of malignancies. EBV is etiologically associated with Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma (NPC) and so on, and KSHV is associated with Kaposi’s sarcoma (KS) (the most common tumor in AIDS patients), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). After infection, gammaherpesviruses can establish acute phase of lytic replication in lungs and then be quickly cleared by the host immune response, while the virus can establish latent infection in spleen through a variety of immune evasion strategies and eventually establish a life-long persistent infection. The presence of latent infection and immune evasion are critical for cells transformation which is closely related to the tumorigenesis.Both EBV and KSHV are highly species-specific viruses and have a very narrow host range, which only infect humans and a few primates. Furthermore, there lacks of cell lines to support efficient de novo productive infection of EBV and KSHV. Mouse infection of a closely related rodent virus, murine herpesvirus 68 (MHV-68) has provided a tractable and valuable in vivo model for elucidating the interaction between gammaherpesviruses and host and evaluating the effectiveness of potential viral vaccines.RTA (replication and transcription activator) serves as a molecular switch for the viral life cycle from latency to lytic replication. It is an immediate-early protein conserved among gammaherpesviruses. RTA is essential for initiating and driving the complete viral life cycle, leading to the release of newly produced viral particles. Studies have shown that continuous expression of RTA can suppress the virus to establish latent infection. ORF73 of MHV-68 can inhibit the expression and function of RTA and disruption of ORF73 compromises the ability of MHV-68 to establish latency. ORF72, M11 and ORF74 are located adjacent to ORF73 on the MHV-68 genome. These genes are transcribed during latency, and closely related to the establishment of latency and tumorigenesis.MHV-68 has adapted several strategies of immune evasion to inhibit the innate and adaptive immune response. ORF10,11,36,54 of MHV-68 can block either the production or signaling of IFNs and M3 is a viral ubiquitin E3 ligase which down-regulates MHC class I expression on the surface of cells. The virus can be easily recognized by the immune system by down-regulation of these genes.Based on the above understandings, we constructed a DIP (deficient in immune evasion and persistence) MHV-68 by overexpressing of RTA, removing of latency-associated proteins (ORF72/73/74, M11) and knocking out of ORF10,11,36, 54 and M3 genes. Mouse experiments showed that DIP virus cannot establish latent infection and can provide immune protection. Therefore, the study of the biological characteristics of the virus and the elicited host immune responses can help us to determine the mechanism of immune regulation and deepen our understandings of latent infection and immune evasion of gammaherpesviruses.The purpose of this thesis is to explore the potential mechanism of latent infection and immune evasion by studying the replication characteristics of DIP in vivo and the induced immune response in the host. And we found DIP infection can induce high production of IFN-y. Therefore, we further clarify the effect of IFN-y on MHV-68 lytic replication, the antagonism of MHV-68 against IFN-y and the cross-talk between type I and type II interferons.Methods and results:In the first section we studied the replication characteristics of DIP in vivo and the induced immune response in the host. We built the in vivo infection model by intranasally infection of BALB/c mice with wild type (WT) or DIP MHV-68, at 3,6 and 14 days post infection infection (dpi), plaque assay or infectious center assay was performed to detect the lytic replication and latent infection of viruses. Results showed that DIP virus cannot establish lytic and latent infection in vivo. The dynamic expressions of viral genes, inflammatory cytokines, interferon-related factors and some cytokines after infection were determined by RT-qPCR. We found that the viral gene transcripts of DIP virus increased rapidly, peaked at day 3 after infection, and then began to decline to a similar level to the WT virus at 6 dpi. Notably, the expression of IFN-y was significantly higher than that of WT virus at 6 dpi. For the inflammatory cytokines IL-1β and TNF-a, they reached a peak at the first day after DIP virus infection, and then gradually decreased to the similar level to the WT virus at 6 dpi. ELISA results showed that DIP can induce high production of IFN-y at 6 dpi in both serum and bronchoalveolar lavage fluid (BALF). These results suggest that IFN-y might play an important role in the anti-viral effect, so we focused on the interaction between IFN-y and virus in the following studies.In the second section, we determined the effect of IFN-y on MHV-68 lytic replication. First we intranasally infected WT and IFN-γR-/- mice with MHV-68, the viral titer and DNA copy number were detected by plaque assay and DNA PCR respectively. Results showed there were no significant differences between WT and IFN-γR-/- mice, so as the viral replication after intraperitoneal infection. Expression and production of IFN-y were detected by RT-qPCR and ELISA after infection in C57BL/6 and BALB/c mice, and there was no detectable IFN-y. Intraperitoneal injection of IFN-y inhibited virus replication as viral titer decreased. Pre-treatment with IFN-y could significantlly inhibit the replication of MHV-68 in BMM, RAW 264.7, MEF, NIH3T3 and MLE-12 cells, shown as the decreased viral titer by plaque assay, the decreased viral DNA copy number by DNA PCR and the decreased expression of ORF50 by RT-qPCR. The inhibitory effect of IFN-y was dependent on the IFN-y receptor since IFN-γ cannot inhibit virus replication in BMMs and PMs from IFN-γR-/- mice.In the third section, we studied the cross-talk between type I and type II interferons. By RT-qPCR and ELISA assay, we found that IFN-y can induce the expression and secretion of type I interferon during MHV-68 infection. Inhibition of IFN-a by neutralizing antibodies led to higher replication of MHV-68. Furthermore, IFN-y could up-regulate the expression of interferon stimulated genes (ISGs), including MX1 and IRF7. After treated with IFN-y, the expression of Toll-like receptors (TLR) on BMMs were detected by RT-qPCR. We found IFN-y can up-regulate TLR9 expression, and the up-regulation depended on IFN-y receptor and STAT1. Western blotting showed after infection, the up-regulation of TLR9 resulted in the enhanced phosphorylation of IRF7. Inhibiting of TLR9 by inhibitor or small interfering RNA, IFN-y induced type I interferon production significantly decreased, accompanied with the increased viral replication. ChIP-qPCR assay result showed that p-STAT1 cannot directly bind to the TLR9 promoter.In the fourth section, we studied the antagonistic effect of virus against IFN-y and identified that MHV-68 infection could induce SOCS1 production which inhibited the anti-viral activity of IFN-y by targeting phosphorylation of STAT1 in BMMs. Firstly, the growth curve of virus was determined, and we found the anti-MHV-68 effect of IFN-y was gradually lost and the phosphorylation of STAT1 was gradually suppressed. By screening of JAK-STAT1 negative regulators by RT-qPCR, we found MHV-68 infection induced SOCS1 expression, while UV-inactivated MHV-68 cannot. BMM cells transfected with SOCS1 specific siRNA can reverse this phenomenon. Inhibition of SOCS1 resulted in persistent anti-viral effect of IFN-y and sustainable phosphorylation of STAT1. Then we determined whether TLRs are involved by knocking down of TLR2, TLR3, TLR4, TLR7 and TLR9 by specific siRNAs, and detected the expression of SOCS1. Results showed that only knocking down of TLR3 significantly reduced MHV-68 stimulated SOCS1 expression. We also found poly (I:C) which is the ligand of TLR3 can induce SOCS1 expression. MyD88 is a key signaling molecule links TLRs signaling other than TLR3. So then we knocked down MyD88 by si-RNA technique in BMMs or used BMM from MyD88’A mice and we found knocking down of MyD88 had no effect on the MHV68 induced SOCS1 expression. We then detected the activation of TLR3 down-streamed signaling pathways by Western blotting after infection. Results showed MHV-68 infection can activate NF-κB, p38, ERK, JNK signaling pathways. With specific inhibitors of these signaling pathways, we determined that SOCS1 induction decreased only after inhibition of NF-κB. Finally, we chose NIH3T3 cells which cannot express TLR3 to check the anti-viral effect and the phosphorylation of STAT1 induced by IFN-y. Strikingly, the viral titer in IFN-y treated group decreased steady in NIH3T3 cells and the phosphorylation of STAT1 stayed steady as well. Overexpression of SOCS1 in NIH3T3 cells attenuated the anti-viral activity of IFN-y, which was consistent with the phosphorylation status of STAT1.Conclusions:1. DIP virus cannot establish lytic replication and latent infection and can induce high production of IFN-y in BALB/c mice, suggesting that IFN-y may play a role in this process and MHV-68 can inhibit the production of IFN-y.2. Receptor-dependent inhibition of lytic replication of MHV-68 in macrophages, fibroblast cells and epithelial cells by IFN-y.3. There exists new mechanism of anti-MHV-68 by IFN-y. By up-regulating the expression of TLR9, IFN-y could enhance the production of type I interferon. Type I and type II interferons synergistically play the anti-MHV68 role.4. MHV-68 infection could induce the expression of cellular factor SOCS1 via activation of TLR3-TRAF-NF-κB, which acts as an antagonism against IFN-y.Innovativenesses:1. Using the murine herpesvirus 68 (MHV-68) as our model, we are the first one to construct a lytic replication, latent infection, immune evasion defective recombinant virus DIP MHV-68. The study of this recombinant virus will help us understand the mechanism of immune protection and the tumorigenesis of gammaherpesviruses such as EBV and KSHV.2. We are the first one to announce that IFN-y could up-regulate TLR9 expression and thus promote type I interferon production. In addition to clarification of the mechanism of inhibition of MHV-68 lytic replication by IFN-y, our study also helps to understand the relationship between type I interferon and type II interferon during the tumor-associated viral infection.3. We clarify that MHV-68 infection in BMMs can induce SOCS1 to inhibit the anti-viral activity of IFN-y. Here we report a new mechanism of how MHV-68 escape from the immune response and provide a new result for understanding the interactions between host and viruses.