Interactions between Viral Regulatory Proteins Ensure an MOI-Independent Probability of Lysogeny during Infection by Bacteriophage P1

Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI.

Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies

ABSTRACTPML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.

Dissecting novel mechanisms of hepatitis B virus-related hepatocellular carcinoma using meta-analysis of public data.

333 Background: Hepatitis B is a cause of hepatocellular carcinoma (HCC) globally, irrespective of viral loads. Interestingly, this process is not necessarily mediated through cirrhosis and may in fact involve oncogenic processes. Prior studies have suggested specific oncogenic gene expression pathways affected by viral regulatory proteins. Thus, identifying these genes and associated pathways could highlight predictive factors for HCC transformation, and has implications in early diagnosis and treatment. Methods: We employed our Search, Tag, Analyze, Resource (STARGEO) platform to conduct a meta-analysis of public data from NCBI's Gene Expression Omnibus. We performed meta-analysis consisting of 155 tumor samples compared against 185 adjacent non-tumor samples. We present the results from the meta-analysis in Ingenuity Pathway Analysis. Results:Our analysis revealed LXR/RXR activation, LPS-IL-1 mediation inhibition of RXR, and FXR/RXR activation as top canonical pathways amongst others. Top upstream regulators identified included the Ras family gene RABL6. The role of RABL6 in oncogenesis is beginning to unfold but its specific role in HBV-related HCC remains undefined. Our causal analysis suggests RABL6 mediates pathogenesis of HBV-related HCC through promotion of genes related to cell division, epigenetic regulation, and Akt signaling. Additionally, HOXA10 was a top upstream regulator and was strongly upregulated in our analysis. While described in other cancer, HOXA10 has not been well documented in HBV-related HCC and our causal analysis suggests it mediates pathogenesis through downregulation of tumor suppressors and its protective effect from oxidative stress. Conclusions: This meta-analysis describes possible role of viral regulatory proteins such as RABL6 and HOXA10 in the pathogenesis of HBV -related HCC. Further prospective studies are needed to confirm our findings.

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral cyclin-dependent kinase ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed; however, the most pronounced pUL97-dependent phenotype could be assigned to viral nuclear egress, as illustrated by deletion of the UL97 gene or pharmacological pUL97 inhibition. Despite earlier data pointing to a cyclin-independent functionality, experimental evidence increasingly emphasized the role of pUL97-cyclin complexes. Consequently, the knowledge about pUL97 involvement in host interaction, viral nuclear egress and additional replicative steps led to the postulation of pUL97 as an antiviral target. Indeed, validation experiments in vitro and in vivo confirmed the sustainability of this approach. Consequently, current investigations of pUL97 in antiviral treatment go beyond the known pUL97-mediated ganciclovir prodrug activation and henceforward include pUL97-specific kinase inhibitors. Among a number of interesting small molecules analyzed in experimental and preclinical stages, maribavir is presently investigated in clinical studies and, in the near future, might represent a first kinase inhibitor applied in the field of antiviral therapy.

Antagonizing the Glucocorticoid Receptor Impairs Explant-Induced Reactivation in Mice Latently Infected with Herpes Simplex Virus 1

ABSTRACTHerpes simplex virus 1 (HSV-1) establishes lifelong latent infections in neurons. Reactivation from latency can lead to serious recurrent disease, including stromal keratitis, corneal scarring, blindness, and encephalitis. Although numerous studies link stress to an increase in the incidence of reactivation from latency and recurrent disease, the mechanism of action is not well understood. We hypothesized that stress, via corticosteroid-mediated activation of the glucocorticoid receptor (GR), stimulates viral gene expression and productive infection during reactivation from latency. Consequently, we tested whether GR activation by the synthetic corticosteroid dexamethasone influenced virus shedding during reactivation from latency using trigeminal ganglion (TG) explants from Swiss Webster mice latently infected with HSV-1, strain McKrae. TG explants from the latently infected mice shed significantly higher levels of virus when treated with dexamethasone. Conversely, virus shedding from TG explants was significantly impaired when they were incubated with medium containing a GR-specific antagonist (CORT-108297) or stripped fetal bovine serum, which lacks nuclear hormones and other growth factors. TG explants from latently infected, but not uninfected, TG contained significantly more GR-positive neurons following explant when treated with dexamethasone. Strikingly, VP16 protein expression was detected in TG neurons at 8 hours after explant whereas infected-cell protein 0 (ICP0) and ICP4 protein expression was not readily detected until 16 hours after explant. Expression of all three viral regulatory proteins was stimulated by dexamethasone. These studies indicated corticosteroid-mediated GR activation increased the number of TG neurons expressing viral regulatory proteins, which enhanced virus shedding during explant-induced reactivation from latency.IMPORTANCEHerpes simplex virus 1 (HSV-1) establishes lifelong latent infections in neurons within trigeminal ganglia (TG); periodically, reactivation from latency occurs, leading to virus transmission and recurrent disease. Chronic or acute stress increases the frequency of reactivation from latency; how this occurs is not well understood. Here, we demonstrate that the synthetic corticosteroid dexamethasone stimulated explant-induced reactivation from latency. Conversely, a glucocorticoid receptor (GR) antagonist significantly impaired reactivation from latency, indicating that GR activation stimulated explant-induced reactivation. The viral regulatory protein VP16 was readily detected in TG neurons prior to infected-cell protein 0 (ICP0) and ICP4 during explant-induced reactivation. Dexamethasone induced expression of all three viral regulatory proteins following TG explant. Whereas the immunosuppressive properties of corticosteroids would facilitate viral spread during reactivation from latency, these studies indicate GR activation increases the number of TG neurons that express viral regulatory proteins during early stages of explant-induced reactivation.

Emerging Role of PML Nuclear Bodies in Innate Immune Signaling

Research in the last 2 decades has demonstrated that a specific organelle of the cell nucleus, termed PML nuclear body (PML-NB) or nuclear domain 10 (ND10), is frequently modified during viral infection. This correlates with antagonization of a direct repressive function of individual PML-NB components, such as the PML, hDaxx, Sp100, or ATRX protein, that are able to act as cellular restriction factors. Recent studies now reveal an emerging role of PML-NBs as coregulatory structures of both type I and type II interferon responses. This emphasizes that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to compromise intrinsic antiviral defense and innate immune responses.

Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses

ABSTRACTPML is the organizer of cellular structures termed nuclear domain 10 (ND10) or PML-nuclear bodies (PML-NBs) that act as key mediators of intrinsic immunity against human cytomegalovirus (HCMV) and other viruses. The antiviral function of ND10 is antagonized by viral regulatory proteins such as the immediate early protein IE1 of HCMV. IE1 interacts with PML through its globular core domain (IE1CORE) and induces ND10 disruption in order to initiate lytic HCMV infection. Here, we investigate the consequences of a point mutation (L174P) in IE1CORE, which was shown to abrogate the interaction with PML, for lytic HCMV infection. We found that a recombinant HCMV encoding IE1-L174P displays a severe growth defect similar to that of an IE1 deletion virus. Bioinformatic modeling based on the crystal structure of IE1COREsuggested that insertion of proline into the highly alpha-helical domain severely affects its structural integrity. Consistently, L174P mutation abrogates the functionality of IE1COREand results in degradation of the IE1 protein during infection. In addition, our data provide evidence that IE1COREas expressed by a recombinant HCMV encoding IE1 1-382 not only is required to antagonize PML-mediated intrinsic immunity but also affects a recently described function of PML in innate immune signaling. We demonstrate a coregulatory role of PML in type I and type II interferon-induced gene expression and provide evidence that upregulation of interferon-induced genes is inhibited by IE1CORE. In conclusion, our data suggest that targeting PML by viral regulatory proteins represents a strategy to antagonize both intrinsic and innate immune mechanisms.IMPORTANCEPML nuclear bodies (PML-NBs), which represent nuclear multiprotein complexes consisting of PML and additional proteins, represent important cellular structures that mediate intrinsic resistance against many viruses, including human cytomegalovirus (HCMV). During HCMV infection, the major immediate early protein IE1 binds to PML via a central globular domain (IE1CORE), and we have shown previously that this is sufficient to antagonize intrinsic immunity. Here, we demonstrate that modification of PML by IE1COREnot only abrogates intrinsic defense mechanisms but also attenuates the interferon response during infection. Our data show that PML plays a novel coregulatory role in type I as well as type II interferon-induced gene expression, which is antagonized by IE1CORE. Importantly, our finding supports the view that targeting of PML-NBs by viral regulatory proteins has evolved as a strategy to inhibit both intrinsic and innate immune defense mechanisms.

Overexpressed NF-κB–inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells

The nuclear factor-κB (NF-κB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-κB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-κB–inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IκBα. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-κB and NF-κB–dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/γcnull mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-κB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Rev Inhibition Strongly Affects Intracellular Distribution of Human Immunodeficiency Virus Type 1 RNAs

ABSTRACT To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.