Interplay of Murine Gammaherpesvirus 68 with NF-kappaB Signaling of the Host

90% similar; triplex-β, 78% identical, 92% similar; small basic capsid protein, 64% identical, 77% similar [W. Transwell migration for HUVEC transduced with 1 MOI lentivirus empty vector (mpCDH) or lentivirus-miR-K6-3p (miR-K6-3p). Sterols and cholesterol were eluted using 70% and 100% ethanol, respectively. 184, 863−871 (1996). Unstained and isotype-staining controls were used for all experiments. This process kills more than 99% of live cells, which allows preformed infectious virus to be discerned from virus reactivating from latently infected cells (58-60). The samples were boiled for 10 min, and equal volumes were loaded onto a 12% Bis-Tris gel.

Second, at least one of the hairpin arms had to harbor a conserved region of at least 6 consecutive nucleotides located within 35 nt of the terminal loop (i.e., the region expected to harbor the mature miRNA). The M2 ISRE binds IRF2 in vivo during latency. The statistical significance of differences observed in viral transcript levels was determined by two-tailed, unpaired t test on background-subtracted phosphorimager volumes. At 4 dpt viral plaques were observed, indicating cytopathic effects; cell-free culture supernatant and the cells were separately harvested. A. Three additional interactions could not be confirmed due to the low expression in our system, but were previously reported between homologous proteins from other herpesvirus family members (Table S1) [29]–[33]. Lysates were centrifuged at 16,000× g for 15 min at 4°C to remove insoluble material.


Homogenization of tissues therefore loses important information. EBV binding/entry rapidly activates STAT3 via Janus kinases. Raw reads were aligned to the RefSeq mRNA database using Bowtie with MAC-like rules. EGFP expression (A), M9 viral … ITC was performed at 25 °C with 25 injections of 2 μl each. SH-SY5Y, human neuroblastoma cells, were infected with MHV-68/EGFP at an MOI of 0.1 or 1 for 72 h. Primary bone marrow macrophages were mock infected or infected at a multiplicity of infection (MOI) of 10 with wild-type or 36-FLAG MHV68 (36F MHV68) (27, 30).

After infection of immunocompetent C57BL/6 mice with each virus, the extent of Vβ4+ CD8+ T cell expansion was assessed at the normal peak of this response at 28 d after infection. (A) BHK-21 cells were infected at a low multiplicity of infection (0.01 PFU/cell) with wild-type, ORF11−STOP, or revertant virus as indicated. Portions of the organ samples were fixed in 10% buffered formalin (Fisher Scientific). Murine gammaherpesvirus 68 (MHV68, formally identified as murid herpesvirus 4) is a natural pathogen of murid rodents used to study virus–host interactions in the context of a whole animal. We previously reported that γHV68 activates the mitochondrion antiviral signaling (MAVS)-IKKβ pathway to promote viral transcriptional activation and lytic infection (10). In addition, the basic biological role of TK in gammaherpesvirus infections is unknown. Aside from the conclusion that a TATT sequence is required, the sequence requirement at each position of the EBV and KSHV late gene core promoters has not been well defined, and the role of individual nucleotides in controlling late gene expression remains to be demonstrated in the context of viral genomes.

This analysis demonstrates that the transcription of gene 50 is enhanced by ongoing viral infection, suggesting that either a newly synthesized viral protein and/or an induced cellular factor augments the transcription of gene 50. RTA binds directly to RTA-responsive elements (RREs) in viral promoters to activate the transcription of viral genes such as PAN, K1, K12, and vIL-6 (9, 10, 22, 52–54), and in these cases, the RREs serve as RTA binding sites (RBSs). It is also documented that CMV stimulates inflammasome activation in a manner that is dependent on AIM2 (4). Therefore, it is important to understand how their lymphocyte infections work. gB, gH, and gL are conserved in all mammalian herpesviruses and are likely to be essential for membrane fusion (9, 23, 25). H. The comparison of these three RTAs has allowed us to determine which domains of RTA are necessary for reactivation of MHV-68 and transactivation of viral lytic promoters.

The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus pose significant health risks worldwide, including the induction of cancer and lymphoproliferative diseases. Expression of the gammaHV68 v-cyclin significantly increased the number of thymocytes in cell culture, as determined by measuring both DNA content and incorporation of 5-bromo-2-deoxyuridine following in vivo pulse-labeling. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8+ T cell response to this protein. We previously showed that, unlike most cell types, ECs survive productive gammaherpesvirus 68 (γHV68) infection and achieve anchorage-independent growth, providing a cellular reservoir for viral persistence. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. Rta, encoded primarily by open reading frame 50, is well conserved among gammaherpesviruses. Gammaherpesviruses characteristically establish latent and persistent infections in their hosts after immunologically mediated clearance of the acute infection.

Interplay of Murine Gammaherpesvirus 68 with NF-kappaB Signaling of the Host

Like other gammaherpesviruses, latent infection of MHV-68 leads to splenomegaly and lymphoproliferative diseases in the host. Concomitant with this lytic infection, the virus establishes latent infection within epithelial and B cells in the lungs (18, 38). Thus, while hypothesis-driven and intuition-based studies have identified many phosphorylation-dependent signaling events that regulate viral replication and host responses to infection, it is likely that the vast majority of infection-associated changes in host protein phosphorylation status are not yet known. E-mail: rsun{at}mednet.ucla.edu. In addition to SARS and Ebola viruses, bats contain and transmit other human pathogens not limited to, but including, Hendra virus (HeV) (9), Nipah virus (NiV) (10), and rabies virus (11, 12). Additionally, we also report infection of neuronal cells by KSHV in vitro similar to that by EBV. KSHV infection is the etiological agent of Kaposi’s sarcoma and is associated with primary effusion lymphoma and multicentric Castleman disease (Verma and Robertson, 2003; Cesarman, 2014).

We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. Lytic replication occurs after de novo infection of a permissive cell or as a result of reactivation of latent virus within a nonpermissive cell. Phone: 49-89-5160-5290. γHV68 has areas of colinear sequence homology with EBV, KSHV, and the primate gammaherpesvirus herpesvirus saimiri (59). Thus, any developing B cell subsets could provide a potential access point for recurrent entry of the virus into the long-lived mature B cell reservoir. Unexpectedly, we found that murine gammaherpesvirus pathogenesis was not enhanced in mice lacking caspase-1, a critical inflammasome component.


Phone: (404) 727-7665. The gammaherpesviruses, including HVS, EBV, KSHV, and RRV, are capable of establishing latent infection in lymphocytes. Here we show that the B cell proliferation driven by a murid γHV requires BAFF-R. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. 1A to C). The presence of Rd2 also led to attenuation of viral lytic protein expression and virion production. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs.

The initiation of a relatively meager pro-inflammatory response during the first few days post-infection has been noted [22], and a listing of HV-68 induced effects on the innate and adaptive immune response has recently been reviewed [9]. Fazakerley, and A. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. There is remarkable functional conservation of muSOX host shutoff activities with those of KSHV SOX, including the recently described ability of SOX to induce mRNA hyperadenylation in the nucleus as well as cause nuclear relocalization of the poly(A) binding protein. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The induction of p53 during MHV68 infection occurred in multiple cell types, including splenocytes of infected mice.

This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Some of these diseases, such as Hodgkin’s and non-Hodgkin lymphoma resemble those occurring in immunocompetent patients, but the proportion of tumors in which EBV is present is increased. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (γHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. Macrophages are the cell type that is responsive to the IFN-γ-mediated control of γHV68 reactivation; however, the molecular mechanism of this IFN-γ action is undefined. The presence of this novel gammaherpesvirus was confirmed by viral metagenomics, while no other viruses other than four novel anelloviruses were detected. In this analysis of chronic infection, we demonstrate that the v-cyclin is required for γHV68-associated mortality in B-cell-deficient mice.

Murine gammaherpesvirus 68 (MHV-68) constitutes the most amenable animal model for this family of pathogens. The mechanisms of latency establishment and maintenance, as well as the switch from latency to lytic replication, are poorly understood. The v-cyclin encoded by murine gammaherpesvirus 68 (γHV68) induces cell cycle progression and is an oncogene (L.