SUMMARY Invading viral DNA can be recognized by the host cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS), resulting in production of the second messenger cGAMP, which directs the adaptor protein STING to stimulate production of type I interferons (IFNs). Although several DNA viruses are sensed by cGAS, viral strategies targeting cGAS are virtually unknown. We report here that Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF52, an abundant gammaherpesvirus-specific tegument protein, subverts cytosolic DNA sensing by directly inhibiting cGAS enzymatic activity through a mechanism involving both cGAS- and DNA-binding. Moreover, ORF52 homologues in other gammaherpesviruses also inhibit cGAS activity and similarly bind cGAS and DNA, suggesting conserved inhibitory mechanisms. Furthermore, KSHV infection evokes cGAS-dependent responses that can limit the infection, and an ORF52-null mutant exhibits increased cGAS signaling. Our findings reveal a mechanism through which gammaherpesviruses antagonize host cGAS DNA sensing.

c‐Jun N‐terminal kinase (JNK) is involved in cancer cell apoptosis; however, emerging evidence indicates that this Janus signaling promotes cancer cell survival. JNK acts synergistically with NF‐κB, JAK/STAT, and other signaling molecules to exert a survival function. JNK positively regulates autophagy to counteract apoptosis, and its effect on autophagy is related to the development of chemotherapeutic resistance. The prosurvival effect of JNK may involve an immune evasion mechanism mediated by transforming growth factor‐β, toll‐like receptors, interferon‐γ, and autophagy, as well as compensatory JNK‐dependent cell proliferation. The present review focuses on recent advances in understanding the prosurvival function of JNK and its role in tumor development and chemoresistance, including a comprehensive analysis of the molecular mechanisms underlying JNK‐mediated cancer cell survival. There is a focus on the specific “Yin and Yang” functions of JNK1 and JNK2 in the regulation of cancer cell survival. We highlight recent advances in our knowledge of the roles of JNK in cancer cell survival, which may provide insight into the distinct functions of JNK in cancer and its potential for cancer therapy.

Fu et al. show that ZMPSTE24 is a broad-spectrum antiviral protein that inhibits entry of selected fusogenic viruses by functioning as an effector in the IFITM pathway. ZMPSTE24 protease activity is dispensable for viral restriction. In mice, ZMPSTE24 deficiency increases susceptibility to influenza infection.

Polymerase basic protein 1 (PB1) is the catalytic core of the influenza A virus (IAV) RNA polymerase complex essential for viral transcription and replication. Understanding the intrinsic mechanisms which block PB1 function could stimulate development of new anti-influenza therapeutics. Affinity purification coupled with mass spectrometry (AP-MS) was used to identify host factors interacting with PB1. Among PB1 interactors, the E3 ubiquitin ligase TRIM32 interacts with PB1 proteins derived from multiple IAV strains. TRIM32 senses IAV infection by interacting with PB1 and translocates with PB1 to the nucleus following influenza infection. Ectopic TRIM32 expression attenuates IAV infection. Conversely, RNAi depletion and knockout of TRIM32 increase susceptibility of tracheal and lung epithelial cells to IAV infection. Reconstitution of trim32-/- mouse embryonic fibroblasts with TRIM32, but not a catalytically inactive mutant, restores viral restriction. Furthermore, TRIM32 directly ubiquitinates PB1, leading to PB1 protein degradation and subsequent reduction of polymerase activity. Thus, TRIM32 is an intrinsic IAV restriction factor which senses and targets the PB1 polymerase for ubiquitination and protein degradation. TRIM32 represents a model of intrinsic immunity, in which a host protein directly senses and counters viral infection in a species specific fashion by directly limiting viral replication.

We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838 -1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wildtype virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication. IMPORTANCEKSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication. K aposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), the most common malignancy in HIV-infected individuals (1, 2). Besides KS, KSHV is associated with two lymphoproliferative diseases, primary effusion lymphoma and multicentric Castleman's disease (3, 4). KSHV belongs to the Gammaherpesvirinae subfamily in the Herpesviridae family and is closely related to rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS), and murine gammaherpesvirus 68 (MHV-68) in the Rhadinovirus genus (␥2). Its closest relative in humans is Epstein-...

The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

Background: ORF45 of Kaposi sarcoma-associated herpesvirus (KSHV) causes sustained activation of p90 ribosomal S6 kinases (RSKs). Results: ORF45 increases phosphorylation of eIF4B through p90 RSKs. Conclusion: The ORF45/RSK axis promotes protein translation during lytic replication. Significance: This mechanism is crucial for understanding of translational regulation during KSHV lytic replication.

Xinjiang, China is an endemic area for Kaposi's sarcoma (KS) but the seroprevalence of Kaposi's sarcoma-associated herpesvirus (KSHV) and risk factors remain undefined. In this study, antibodies to one KSHV latent protein (ORF73) and two KSHV lytic proteins (ORF65 and ORF-K8.1) were examined in 2,228 subjects from the general population and 37 subjects infected with HIV-1 in Xinjiang, and 560 subjects from the general population in Hubei, a low KS incidence region. The serostatus of a serum sample was defined based on positive results in any one of the three serologic assays. The seroprevalence of KSHV in the general population was higher in Xinjiang than in Hubei (19.2% vs 9.5%; odds ratios [OR], 2.28; 95% confidence interval [CI], 1.68-3.08; P < 0.001). Among the ethnic groups in Xinjiang, 68 (15.8%) Han, 182 (20.7%) Uygur, 140 (19.9%) Hazakh, 9 (33.3%) Xibo, and 29 (16.8%) Hui were KSHV-seropositive, respectively. Compared to the Han, the latter groups had an increase in the risk of KSHV of 62.2%, 63.8%, 180.1% and 30.2% (P = 0.003, 0.004, 0.018, and 0.286, respectively). Subjects aged < 20, 20-50, and > 50 had a seroprevalence of KSHV of 11.8%, 17.9% and 24.6%, respectively. Compared to subjects aged < 20, the latter groups had an increase in the risk of KSHV of 63.3% and 144.5% (P = 0.009 and < 0.001, respectively). Subjects infected with HIV-1 in Xinjiang had a seroprevalence of KSHV of 43.2%, and a 220% increase in the risk of KSHV compared to the general population (P < 0.001). Similar results were obtained when the seroprevalence of KSHV was analyzed with any single or two of the three serologic assays alone. Genotyping identified 3 unique sequences clustered in the A clade. This study indicates that Xinjiang has a high seroprevalence of KSHV. Geographic location, ethnicity, age and HIV-1 infection are risk factors. Serologic and genotyping results suggest the introduction of KSHV into Xinjiang by specific ethnic groups.