cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection.

Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.

Relative Contributions of Herpes Simplex Virus 1 ICP0 and vhs to Loss of Cellular IFI16 Vary in Different Human Cell Types.

UNLABELLED: The herpes simplex virus 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase that promotes the degradation of several host cell proteins. Most studies have found that ICP0 promotes the loss of IFI16 in infected cells, but one study reported that ICP0 was not necessary or sufficient for loss of IFI16 in a tumor-derived cell line. Therefore, in this study, we examined the requirement for ICP0 in promoting the loss of IFI16 in several normal and tumor-derived cell lines. HSV-1 infection resulted in an observable decrease of IFI16 protein levels in normal human foreskin fibroblasts (HFFs), normal oral keratinocytes (NOKs), and HeLa cells but not in U2OS cells. During infection with an ICP0-null virus, we observed a reduced loss of IFI16 in HFFs and NOKs but not in HeLa cells. Ectopic expression of ICP0 from a transfected plasmid was sufficient to promote the loss of IFI16 in HFFs and NOKs. In the absence of ICP0, we observed a delayed reduction of IFI16 protein that correlated with a reduction in the steady-state levels of IFI16 mRNA. In addition, we show that the ICP0-independent loss of IFI16 in HeLa cells is dependent in part on the activity of the viral virion host shutoff (vhs) tegument protein. Together, these results demonstrate that HSV-1 promotes the loss of IFI16 through at least two mechanisms: (i) by ICP0-dependent degradation of IFI16 and (ii) by vhs-dependent turnover of IFI16 mRNA. In addition, this study highlights a potential intrinsic difference between normal and tumor-derived cells for the activities of IFI16 and HSV-1 ICP0. IMPORTANCE: HSV-1 is a ubiquitous virus that establishes a lifetime persistent infection in humans. The relative success of HSV-1 as a pathogen is, in part, dependent on the expression of viral proteins that counteract host intrinsic defense mechanisms and that modulate immune responses during viral infection. In this study, we examined the relative roles of two viral gene products for the ability to promote loss of the antiviral IFI16 DNA sensor. We demonstrate that the viral immediate early ICP0 protein plays a dominant role in the loss of IFI16 in normal, but not tumor-derived, human cell lines. In contrast, viral vhs-mediated loss of IFI16 by mRNA destabilization is revealed to be dominant in tumor-derived cells in which ICP0 is nonfunctional. Together, these results contribute to our understanding of how HSV-1 modulates IFI16 protein levels and highlight cell-type-dependent differences between normal and tumor-derived cells.

miRNA regulation of BK polyomavirus replication during early infection.

Viral microRNAs (miRNAs) play an important role during infection by posttranscriptionally regulating both host and viral gene expression. However, the function of many viral miRNAs remains poorly understood. In this study, we investigated the role of the BK polyomavirus (BKPyV) miRNA in regulating virus replication. The function of the polyomavirus miRNA was investigated in archetype BKPyV, which is the transmissible form of the virus and thought to establish a persistent infection in the host urinary tract. In agreement with previous studies, we show that the BKPyV miRNA targets early mRNAs. Importantly, we show that the miRNA plays a significant role in limiting archetype BKPyV replication in a natural host cell model of infection. This regulation is accomplished through the balance of regulatory elements located within the noncoding control region that control early gene expression and miRNA expression before genome replication. We therefore provide evidence for a unique function of the polyomavirus miRNA that may have important implications for the mechanism of viral persistence.

Herpes simplex virus infected cell protein 8 is required for viral inhibition of the cGAS pathway.

DNA virus infection triggers an antiviral type I interferon (IFN) response in cells that suppresses infection of surrounding cells. Consequently, viruses have evolved mechanisms to inhibit the IFN response for efficient replication. The cellular cGAS protein binds to double-stranded DNA and synthesizes the small molecule cGAMP to initiate DNA-dependent type I IFN production. We showed previously that cGAMP production is relatively low during HSV-1 infection compared to plasmid DNA transfection. Therefore, we hypothesized that HSV-1 produces antagonists of the cGAS DNA sensing pathway. In this study, we found that the HSV-1 ICP8 protein is required for viral inhibition of the cGAS pathway by reducing cGAMP levels stimulated by double-stranded DNA transfection. ICP8 alone inhibited the cGAMP response and may inhibit cGAS action by direct interaction with DNA, cGAS, or other infected cell proteins. Our results reveal another cGAS antiviral pathway inhibitor and highlight the importance of countering IFN for efficient viral replication.

Comparison of cefoxitin and oxacillin disk diffusion methods for detection of mecA-mediated resistance in Staphylococcus aureus in a large-scale study.

The recommended breakpoints for the cefoxitin disk diffusion test for Staphylococcus aureus were recently modified. In this large-sample study, cefoxitin sensitivity and specificity compared to those of oxacillin were 97.3% and 100%, respectively. This study validated the new cefoxitin breakpoints for the detection of mecA-mediated resistance in S. aureus.

ABIN-1 heterozygosity sensitizes to innate immune response in both RIPK1-dependent and RIPK1-independent manner.

ABIN-1 (encoded by the gene Tnip1) is a ubiquitin-binding protein that can interact with ubiquitin-editing enzyme A20 (encoded by the gene TNFAIP3) to restrain the activation of necroptosis and NF-κB activation. Genetic variants in the genes Tnip1 and TNFAIP3 are both strongly associated with susceptibility to autoimmune chronic inflammatory diseases such as psoriasis vulgaris and systemic lupus erythematosus (SLE) in humans. Here we investigated the mechanism by which ABIN-1 regulated innate immune responses. We show that ABIN-1 heterozygosity sensitizes cells to antiviral response by mediating NF-κB-dependent and RIPK1-independent expression of pattern recognition molecules, including TLR3, RIG-I, and MDA5, in MEFs. Furthermore, we demonstrate that increased interaction of ABIN-1 and A20 with prolonged poly(I:C) stimulation of WT cells leads to A20-dependent reduction of ABIN-1 protein. Finally, we show that ABIN-1 heterozygosity sensitizes innate immune response of Abin-1+/- mice in vivo by promoting the production of proinflammatory cytokines, which can be blocked upon inhibition of RIPK1 kinase. Inhibition of RIPK1 kinase activity in vivo partially reduces the expression of MDA5, RIG-I, and caspase-11 in Abin-1+/- mice but not in WT mice. Thus, we conclude that ABIN-1 is a suppressor of innate immune response and the interaction of ABIN-1 with A20 controls innate immunity response through the NF-κB pathway and in both RIPK1 kinase activity-independent and dependent manner.

A Mouse Model of Multi-Drug Resistant Staphylococcus aureus-induced Ocular Disease.

Staphylococcus aureus infection of the cornea is a significant threat to vision. The percentage of bacterial isolates resistant to antibiotics is increasing as is the percentage of infections caused by methicillin resistant isolates. There is a critical need for additional therapeutic approaches and their development will require the use of animal models to test efficacy. Two mouse models of S. aureus keratitis have been described but only quantified stromal keratitis (corneal clouding and perforation). We have extended these models using the methicillin resistant S. aureus USA300 LAC strain and show that eyelid inflammation and swelling (blepharitis) and corneal neovascularization can be quantified. This expanded model should prove useful in assessing additional effects of antibacterial therapies and additional pathological mechanisms involved in bacterial ocular infection.

Efficient propagation of archetype BK and JC polyomaviruses.

BKPyV and JCPyV are closely related, ubiquitous human pathogens that cause disease in immunocompromised patients. The DNA sequence of the regulatory regions distinguishes two forms of these viruses, designated archetype and rearranged. Although cell culture systems exist for rearranged BKPyV and JCPyV, currently there is no robust cell culture system to study the archetype viruses. Large T antigen (TAg) is a virally encoded protein required to initiate viral DNA synthesis. Because archetype virus produces undetectable levels of TAg, we hypothesized that TAg overexpression would stimulate archetype virus replication. Efficient propagation of the archetype forms of BKPyV and JCPyV was observed in 293TT cells, human embryonic kidney cells overexpressing SV40 TAg. Importantly, the archetypal structure of the regulatory region was maintained during viral growth. Significant replication was not observed for Merkel cell, KI, or WU polyomaviruses. 293TT cells provide a means of propagating archetype BKPyV and JCPyV for detailed study.

BK polyomavirus: emerging pathogen.

BK polyomavirus (BKPyV) is a small double-stranded DNA virus that is an emerging pathogen in immunocompromised individuals. BKPyV is widespread in the general population, but primarily causes disease when immune suppression leads to reactivation of latent virus. Polyomavirus-associated nephropathy and hemorrhagic cystitis in renal and bone marrow transplant patients, respectively, are the most common diseases associated with BKPyV reactivation and lytic infection. In this review, we discuss the clinical relevance, effects on the host, virus life cycle, and current treatment protocols.

A system for the analysis of BKV non-coding control regions: application to clinical isolates from an HIV/AIDS patient.

The human polyomavirus BK virus (BKV) is an important opportunistic pathogen whose disease prevalence continues to increase with the growing immunocompromised population. To date, the major determinant of replication in cell culture has not been formally proven. BKV exists as archetype virus and rearranged variants, which are classified based on the DNA sequence of their non-coding control regions (NCCRs). The archetype BKV NCCR is divided into five blocks of sequence and rearranged variants contain deletions and duplications of these blocks. In this study, a genetic system was developed and used to identify the major determinant of replication ability in primary renal proximal tubule epithelial cells, the natural host cell of BKV. This system was also used to analyze NCCR variants isolated from an immunocompromised patient which contain assorted rearrangement patterns and functional differences. This study solidifies the NCCR as the major genetic determinant of BKV replication ability in vitro.