Regulation Of Viral Replication Research Articles

IL-27, a Cytokine, and IFN-λ1, a Type III IFN, Are Coordinated To Regulate Virus Replication through Type I IFN

IL-27, a member of the IL-12 family, plays a critical role in the control of innate and adaptive immune responses. IFN-λ1, a member of the type III IFN family, shows antiviral abilities. In this study, we investigated the effects of IL-27 and IFN-λ1 on the replication of hepatitis B virus (HBV), a major pathogen associated with a high risk for cirrhosis, liver failure, and hepatocellular carcinoma. We revealed that HBV infection activates IL-27 expression and IFN-λ1 production and demonstrated that viral-activated IL-27 and IFN-λ1 are coordinated to inhibit HBV replication. Initially, HBV infection upregulates IL-27 expression, which, in turn, stimulates IFN-λ1 production through regulating ERK1/2 signaling and by enhancing NF-κB nuclear translocation to bind to the IFN-λ1 promoter. Moreover, IL-27-activated IFN-λ1 upregulates IFN-λ1 receptor (IL-28R1 and IL-10Rβ) activity, resulting in the activation of the STAT1/2 pathway, which, in turn, induces the expression of IFN-stimulated genes, including IFN-inducible dsRNA-activated protein kinase, oligoadenylate synthetase 1, and IFN-induced GTP-binding protein 1 and, finally, inhibits HBV protein expression and viral capsid-associated DNA replication. More interestingly, we also revealed that type I IFN (IFN-α) is also involved in the downregulation of HBV replication mediated by IL-27. Thus, we identified a previously unknown mechanism by which IL-27 and IFN-λ1 are coordinated to regulate virus replication through type I IFN.

RNA Viruses: ROS-Mediated Cell Death.

Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections.

Antiviral effects of inhibiting host gene expression.

RNA interference (RNAi) has been used to probe the virus-host interface to understand the requirements for host-gene expression needed for virus replication. The availability of arrayed siRNA libraries has enabled a genome-scale, high-throughput analysis of gene pathways usurped for virus replication. Results from these and related screens have led to the discovery of new host factors that regulate virus replication. While effective delivery continues to limit development of RNAi-based drugs, RNAi-based genome discovery has led to identification of druggable targets. These validated targets enable rational development of novel antiviral drugs, including the rescue and repurposing of existing, approved drugs. Existing drugs with known cytotoxicity and mechanisms of action can potentially be re-targeted to regulate host genes and gene products needed by influenza to replicate. Drug repositioning is more cost-effective, less time-consuming, and more effective for anti-influenza virus drug discovery than traditional methods. In this chapter, a general overview of RNAi screening methods, host-gene discovery, and drug repurposing is examined with emphasis on utilizing RNAi to identify druggable genes that can be targeted for drug development or repurposing.

Global Reprogramming of the Cellular Translational Landscape Facilitates Cytomegalovirus Replication

Unlike many viruses that suppress cellular protein synthesis, host mRNA translation and polyribosome formation are stimulated by human cytomegalovirus (HCMV). How HCMV impacts the translationally regulated cellular mRNA repertoire and its contribution to virus biology remains unknown. Using polysome profiling, we show that HCMV presides over the cellular translational landscape, selectively accessing the host genome to extend its own coding capacity and regulate virus replication. Expression of the HCMV UL38 mTORC1-activator partially recapitulates these translational alterations in uninfected cells. The signature of cellular mRNAs translationally stimulated by HCMV resembles pathophysiological states (such as cancer) where translation initiation factor levels or activity increase. In contrast, cellular mRNAs repressed by HCMV include those involved in differentiation and the immune response. Surprisingly, interfering with the virus-induced activation of cellular mRNA translation can either limit or enhance HCMV growth. The unanticipated extent to which HCMV specifically manipulates host mRNA translation may aid in understanding its association with complex inflammatory disorders and cancer.

HSP90 Protects the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Oncoprotein from Proteasomal Degradation To Support NF-κB Activation and HTLV-1 Replication

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients.

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.

The Schlafen Family of Proteins and Their Regulation by Interferons

The Schlafen (SLFN) family of proteins includes several mouse and human members. There is emerging evidence that members of this family of proteins are involved in important functions, such as the control of cell proliferation, induction of immune responses, and the regulation of viral replication. These proteins span across all species with great diversity, with 10 murine and 5 human isoforms. Recent work has established that mouse and human SLFN proteins are regulated by interferons (IFNs). Several Slfn genes were shown to be induced as classical interferon-stimulated genes, and emerging evidence suggests that these proteins play important roles in the growth inhibitory and antineoplastic effects of IFNs. In the current review, the known properties of mouse and human SLFNs are reviewed, and the implications of their emerging functions are discussed.

Systems Biology Analysis of HIV‐Host Interactions

Viruses like HIV‐1, the cause of AIDS must exploit numerous cellular functions to facilitate their replication. At the same time they must counteract both innate and adaptive host immune responses to avoid virus clearance. Along with our collaborators we are using systems biology approaches to dissect the networks of cellular cofactors and restriction factors that regulate virus replication. These studies have revealed a novel role for the cellular sulfonation pathway in regulating HIV‐1 gene expression, numerous novel cellular substrates for HIV‐1 accessory factors, and has revealed a new role for viral apoptotic mimicry in regulating the cellular antiviral response. Our progress in these areas will be discussed.

Role and application of RNA interference in replication of influenza viruses

RNA interference (RNAi) is a naturally occurring endogenous biological post-transcriptional cellular mechanism that regulates RNA expression. Small interfering RNAs (siRNAs), mediators of RNAi, are short (19-26nt), double-stranded RNA duplexes that inhibit gene expression by inducing sequence-specific degradation of homologous messenger RNA (mRNA). Influenza virus infection is a major public health problem, causing hundreds of thousands of deaths worldwide each year. RNAi have provided a means to performing genome-wide screens to determine and validate host cell genes that may be required for influenza replication and treatment with siRNA specific to regions of the influenza genes which can inhibit influenza virus replication. Moreover, influenza virus is using small RNA in the regulation of virus replication and in modulation of the innate immune signalling system of the host. influenza virus; RNA interference; siRNA; miRNA; replication; transcription.

Porcine circovirus type 2 (PCV2): pathogenesis and interaction with the immune system.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). The virus preferentially targets the lymphoid tissues, which leads to lymphoid depletion and immunosuppression in pigs. The disease is exacerbated by immunostimulation or concurrent infections with other pathogens. PCV2 resides in certain immune cells, such as macrophage and dendritic cells, and modulates their functions. Upregulation of IL-10 and proinflammatory cytokines in infected pigs may contribute to pathogenesis. Pig genetics influence host susceptibility to PCV2, but the viral genetic determinants for virulence remain unknown. PCV2 DNA and proteins interact with various cellular genes that control immune responses to regulate virus replication and pathogenesis. Both neutralizing antibodies and cell-mediated immunity are important immunological correlates of protection. Despite the availability of effective vaccines, variant strains of PCV2 continue to emerge. Although tremendous progress has been made toward understanding PCV2 pathogenesis and immune interactions, many important questions remain.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1α and PKR

BackgroundInteraction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1α (ck1α) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV.ResultsSerine 232 of NS5A is known to be phosphorylated by human ck1α. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1α has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1α has been identified from the model and these are found to be conserved well in the ck1 family. ck1α – substrate peptide complex has also been used to understand the structural basis of association between ck1α and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available.Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified.ConclusionsThe substrate interacting residues in ck1α have been identified using the structural model of kinase - substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection

Toll-like receptor 7 (TLR7) recognizes guanidine-rich viral ssRNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs such as the central nervous system (CNS) is not as clear. This study examined the influence of TLR7 on the neurovirulence of Langat virus (LGTV), a ssRNA tick-borne flavivirus. TLR7 deficiency did not substantially alter the onset or incidence of LGTV-induced clinical disease; however, it did significantly affect virus levels in the CNS with a log(10) increase in virus titres in brain tissue from TLR7-deficient mice. This difference in virus load was also observed following intracranial inoculation, indicating a direct effect of TLR7 deficiency on regulating virus replication in the brain. LGTV-induced type I interferon responses in the CNS were not dependent on TLR7, being higher in TLR7-deficient mice compared with wild-type controls. In contrast, induction of pro-inflammatory cytokines including tumour necrosis factor, CCL3, CCL4 and CXCL13 were dependent on TLR7. Thus, although TLR7 is not essential in controlling LGTV pathogenesis, it is important in controlling virus infection in neurons in the CNS, possibly by regulating neuroinflammatory responses.

Poly(A)-binding protein interacts with the nucleocapsid protein of porcine reproductive and respiratory syndrome virus and participates in viral replication

Interactions between host factors and the viral protein play important roles in host adaptation and regulation of virus replication. Poly(A)-binding protein (PABP), a host cellular protein that enhances translational efficiency by circularizing mRNAs, was identified by yeast two-hybrid screening as a cellular partner for PRRSV nucleocapsid (N) protein in porcine alveolar macrophages. The specific interaction of PRRSV N protein with PABP was confirmed in infected cells by co-immunoprecipitation and in vitro by GST pull-down assay. We showed by confocal microscopy that the PABP co-localized with the PRRSV N protein. Using a series of deletion mutants, the interactive domain of N protein with PABP was mapped to a region of amino acids 52–69. For PABP, C-terminal half, which interestingly interacts other translation regulators, was determined to be the domain interactive with N protein. Short hairpin RNA (shRNA)-mediated silencing of PABP in cells resulted in significantly reduced PRRSV RNA synthesis, viral encoded protein expression and viral titer. Overall, the results presented here point toward an important role for PABP in regulating PRRSV replication.

ZBRK1 represses HIV-1 LTR-mediated transcription

The identification of cellular proteins that interact with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) provides a basic understanding of HIV-1 gene expression, which is the major determinant regulating virus replication. We show that ZBRK1 negatively regulates the HIV-1 LTR. Ectopic expression of ZBRK1 represses transcriptional activity of the HIV-1 LTR, whereas the depletion of endogenous ZBRK1 leads to activation of the HIV-1 LTR. The repressor activity of ZBRK1 is required for TRIM28 binding. Furthermore, ZBRK1 is bound to the HIV-1 LTR in vivo. These results indicate that ZBRK1 could be involved in a potent intrinsic antiretroviral defense. Structured summary of protein interactionsZBRK1physically interacts with TRIM28 by anti tag coimmunoprecipitation (View interaction).

The p53 target gene TRIM22 directly or indirectly interacts with the translation initiation factor eIF4E and inhibits the binding of eIF4E to eIF4G

Background informationThe interferon (IFN)‐inducible protein TRIM22 (Staf50) is a member of the tripartite motif protein family and has been suggested a role in the regulation of viral replication as well as of protein ubiquitylation. In addition, we have previously shown that TRIM22 is a direct target gene for the tumour suppressor p53. Consistently, over‐expression of TRIM22 inhibits the clonogenic growth of monoblastic U937 cells, suggesting anti‐proliferative or cell death‐inducing effects.ResultsHere, we demonstrate that TRIM22 directly or indirectly interacts with the eukaryotic translation initiation factor (eIF)4E, and inhibits the binding of eIF4E to eIF4G, thus disturbing the assembly of the eIF4F complex, which is necessary for cap‐dependent translation. Furthermore, TRIM22 exerts a repressive effect on luciferase reporter protein levels and to some extent on radiolabelled methionine incorporation. Even though all nuclear mRNAs are capped, some are more dependent on eIF4F than others for translation. The translation of one of these mRNAs, IRF‐7C, was indeed found to be repressed in the presence of TRIM22.ConclusionsOur data suggest TRIM22 to repress protein translation preferably of some specific mRNAs. Taken together, we show that TRIM22 represses translation by inhibiting the binding of eIF4E to eIF4G, suggesting a mechanism for regulation of protein translation, which may be of importance in response to p53 and/or IFN signalling.

Whole genome scan identifies several chromosomal regions linked to equine sarcoids

Despite the evidence for a genetic predisposition to develop equine sarcoids (ES), no whole genome scan for ES has been performed to date. The objective of this explorative study was to identify chromosome regions associated with ES. The studied population was comprised of two half-sibling sire families, involving a total of 222 horses. Twenty-six of these horses were affected with ES. All horses had been previously genotyped with 315 microsatellite markers. Quantitative trait locus (QTL) signals were suggested where the F statistic exceeded chromosome-wide significance at P < 0.05. The QTL analyses revealed significant signals reaching P < 0.05 on equine chromosome (ECA) 20, 23 and 25, suggesting a polygenic character for this trait. The candidate regions identified on ECA 20, 23 and 25 include genes regulating virus replication and host immune response. Further investigation of the chromosome regions associated with ES and of genes potentially responsible for the development of ES could form the basis for early identification of susceptible animals, breeding selection or the development of new therapeutic targets.

Behaviour of influenza A viruses differentially expressing segment 2 gene products in vitro and in vivo

The influenza A virus genome comprises eight segments of negative-sense RNA that encode up to 12 proteins. RNA segment 2 encodes three proteins, PB1, PB1-F2 and N40, that are translated from the same mRNA by ribosomal leaky scanning and reinitiation. PB1 is a subunit of the trimeric viral RNA polymerase. PB1-F2 has been reported to be a potential virulence factor, and has been shown to be involved in a number of activities including induction of apoptosis, regulation of virus replication and modulation of the immune response. No function has yet been ascribed to N40, which represents an N-terminally deleted form of PB1. Previous studies on PB1-F2 function mainly used viruses genetically engineered to prevent PB1-F2 expression by mutation of the PB1-F2 start codon. However, ablation of the start codon was shown to increase the expression level of the downstream protein N40. In the present study, we generated recombinant A/WSN/33 viruses carrying different combinations of PB1-F2- and N40-knockout mutations. Overexpression of N40 in a PB1-F2-deficient background had a detrimental effect on virus growth in vitro and in vivo. However, ablation of PB1-F2 or N40 expression individually was not disadvantageous for the virus. Primer-extension analyses revealed an increase in vRNA production by viruses that overexpressed N40. Our data suggest that the observed attenuation of mutant viruses in vitro and in vivo results from these changes in transcription and replication.

Role of the p21-activated kinases (PAKs) in influenza A virus replication

Influenza A virus infection stimulates a wide range of virus-supportive or antiviral mechanisms in host cells. p21-Activated kinase 1 (PAK1) is a serine/threonine kinase that regulates a number of fundamental cellular processes and has been implicated in the modulation of virus replication. Here, we investigated the role of PAK1 activation during influenza A virus infection and found that virus propagation corresponded to stimulated PAK1 phosphorylation. Moreover, transfection of the active form of PAK1 (PAK1-T423E) in A549 cells induced higher viral titers (∼10-fold differences) compared to that in the control vector or inactive PAK1 (PAK1-K299R)-transfected cells. PAK1-specific siRNA knockdown also resulted in 10–100-fold reductions in virus yields compared to that in the control siRNA-treatment (p<0.05). We further showed that treatment with PAK18, a PAK1 peptide inhibitor, resulted in marked suppression of both ERK 1/2 phosphorylation and infectious virus production, which was comparable to that by U0126, a specific MEK/ERK inhibitor. These results provide evidence for the importance of PAK1 activation during influenza virus infection and its association with ERK in regulating virus replication. The present study also implicates PAK1 as a potential therapeutic target for managing influenza virus infections.

Macrophages and HIV-1

Macrophages play an important role in HIV-1 pathogenesis and contribute to the establishment of the viral reservoir responsible for continuous virus production. This review will discuss new insights into HIV-1 infection in macrophages and the effect of infection on immune function and pathology. New cellular factors interacting with various steps of the HIV-1 replication cycle, such as entry, integration, transcription, and assembly of new viral progeny, have been identified. Cellular and viral microRNAs have been shown to regulate virus replication, promote viral latency, and prolong cell survival. Interference with innate immune functions, like phagocytosis, autophagy, cytokine production, and T-cell activation by HIV-1 has been found to contribute to virus replication and latency. Growing evidence indicates an important role of infected macrophages in a variety of HIV-1-associated diseases, including neurocognitive disorders. Under combined antiretroviral therapy (cART), HIV-1 continues to persist in macrophages. Better understanding of HIV-1 infection in macrophages may lead to new adjunctive therapies to improve cART, specifically targeting the viral reservoir and ameliorating tissue-specific diseases.

Preferential Induction of Protective T Cell Responses to Theiler's Virus in Resistant (C57BL/6 × SJL)F1 Mice

Infection of the central nervous system (CNS) with Theiler's murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2(s)) but not in strains such as C57BL/6 (H-2(b)). In addition, it has been shown that (C57BL/6 × SJL/J)F1 mice (F1 mice), which carry both resistant and susceptible MHC haplotypes (H-2(b/s)), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying the resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2(b)-restricted CD8(+) T cell responses than of the H-2(s)-restricted CD8(+) T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice than in susceptible SJL/J mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulation of viral replication and induction of protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis.