The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.

Infectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-Å-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) viruses.IMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages.

Control of the negative IRES trans-acting factor KHSRP by ubiquitination.

Cells and viruses can utilize internal ribosome entry sites (IRES) to drive translation when cap-dependent translation is inhibited by stress or viral factors. IRES trans-acting factors (ITAFs) are known to participate in such cap-independent translation, but there are gaps in the understanding as to how ITAFs, particularly negative ITAFs, regulate IRES-driven translation. This study found that Lys109, Lys121 and Lys122 represent critical ubiquitination sites for far upstream element-binding protein 2 (KHSRP, also known as KH-type splicing regulatory protein or FBP2), a negative ITAF. Mutations at these sites subsequently reduced KHSRP ubiquitination and abolished its inhibitory effect on IRES-driven translation. We further found that interaction between the Kelch domain of Kelch-like protein 12 (KLHL12) and the C-terminal domain of KHSRP contributed to KHSRP ubiquitination, leading to downregulation of enterovirus IRES-mediated translation in infected cells and increased competition against other positive ITAFs. Together, these results show that ubiquitination can exert control over IRES-driven translation via modification of ITAFs, and to the best of our knowledge, this is the first description of such a regulatory mechanism for IRES-dependent translation.

Selection of Domestic Cell Lines for the Creation of Diagnostic and Preventive Preparations against Enterovirus 71.

We studied the sensitivity of domestic proprietary human and animal cell lines from the collection of M. P. Chumakov Federal Scientific Center for Research and Development of Immuneand-Biological Products to infection with different enterovirus 71 strains. A cell system based on domestic proprietary permanent cell line 4647 was for the first time used for reproduction of four enterovirus 71 strains (BrCr, 42266, 42934, and 43374). It was shown that strain 4647 is the optimal cell substrate for enterovirus 71 reproduction. The titers of enterovirus 71 for all four strains considerably (by 2 lgTCID50/ml and more) increased during sequential passages in permanent cell line 4647. The prospects of using permanent cell line 4647 for creation of diagnostic and preventive preparations against 71 was demonstrated.

The 3C protease of enterovirus A71 counteracts the activity of host zinc-finger antiviral protein (ZAP).

Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.

Cell therapy could be a potential way to improve lipoprotein lipase deficiency.

BACKGROUND: Lipoprotein lipase (LPL) deficiency is an autosomal recessive genetic disorder characterized by extreme hypertriglyceridemia, with no cure presently available. The purpose of this study was to test the possibility of using cell therapy to alleviate LPL deficiency. METHODS: The LPL coding sequence was cloned into the MSCV retrovirus vector, after which MSCV-hLPL and MSCV (empty construct without LPL coding sequence) virion suspensions were made using the calcium chloride method. A muscle cell line (C2C12), kidney cell line (HEK293T) and pre-adipocyte cell line (3 T3-L1) were transfected with the virus in order to express recombinant LPL in vitro. Finally, each transfected cell line was injected subcutaneously into nude mice to identify the cell type which could secret recombinant LPL in vivo. Control cells were transfected with the MSCV empty vector. LPL activity was analyzed using a radioimmunoassay. RESULTS: After virus infection, the LPL activity at the cell surface of each cell type was significantly higher than in the control cells, which indicates that all three cell types can be used to generate functional LPL. The transfected cells were injected subcutaneously into nude mice, and the LPL activity of the nearby muscle tissue at the injection site in mice injected with 3 T3-L1 cells was more than 5 times higher at the injection sites than at non-injected control sites. The other two types of cells did not show this trend. CONCLUSION: The subcutaneous injection of adipocytes overexpressing LPL can improve the LPL activity of the adjacent tissue of nude mice. This is a ground-breaking preliminary study for the treatment of LPL deficiency, and lays a good foundation for using cell therapy to correct LPL deficiency.

Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness.

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-ß) gene (Ifn-ß) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Human papillomavirus-associated head and neck squamous cell carcinoma cells lose viability during triggered myocyte lineage differentiation.

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease, and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed, and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells, we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions, including irregularly enlarged cell morphology, cell cycle arrest with downregulation of Ki67, and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation, and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable.

Striated muscle involvement in experimental oral infection by herpes simplex virus type 1.

Herpes simplex virus type 1 is one of the most frequent causes of oral infection in humans, especially during early childhood. Several experimental models have been developed to study the pathogenesis of this virus but all of them employed adult animals. In this work, we developed an experimental model that uses mice younger than 4 days old, to more closely resemble human infection. Mice were infected subcutaneously with the prototype strain McIntyre of Herpes simplex-1, and the progression of infection was studied by immunoperoxidase. All animals died within 24-72 h post-infection, while viral antigens were found in the oral epithelium, nerves and brain. The most striking result was the finding of viral antigens in the nucleus and cytoplasm of cells belonging to striated muscles. Organotypic cultures of striated muscles were performed, and viral replication was observed in them by immunocytochemistry, electron microscopy and viral isolation. We conclude that the infection of striated muscles is present from the onset of oral infection and, eventually, could explain some clinical observations in humans.

Human rhinovirus 14 enters rhabdomyosarcoma cells expressing icam-1 by a clathrin-, caveolin-, and flotillin-independent pathway.

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na(+)/H(+) ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via alpha(v) integrin/CD46 in HeLa cells.

Differentiation-dependent susceptibility of human muscle cells to Zika virus infection.

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.

EV71 3C protease induces apoptosis by cleavage of hnRNP A1 to promote apaf-1 translation.

Enterovirus 71 (EV71) induces apoptosis to promote viral particle release. Earlier work showed that EV71 utilizes its 3C protease to induce apoptosis in a caspase-3-dependent pathway, though the mechanism is unknown. However, work from Vagner, Holcik and colleagues showed that host protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds the IRES of cellular apoptotic peptidase activating factor 1 (apaf-1) mRNA to repress its translation. In this work, we show that apaf-1 expression is essential for EV71-induced apoptosis. EV71 infection or ectopic expression of 3C protease cleaves hnRNP A1, which abolishes its binding to the apaf-1 IRES. This allows IRES-dependent synthesis of apaf-1, activation of caspase-3, and apoptosis. Thus, we reveal a novel mechanism that EV71 utilizes for virus release via a 3C protease-hnRNP A1-apaf-1-caspase-3-apoptosis axis.

Novel role for integrin-linked kinase in modulation of coxsackievirus B3 replication and virus-induced cardiomyocyte injury.

Viral myocarditis is a major cause of sudden cardiac death in children and young adults. Among viruses, coxsackievirus B3 (CVB3) is the most common agent for myocarditis. Recently, more consideration has been given to the role of signaling pathways in pathogenesis of enteroviral myocarditis, providing new platform for identifying a new potential therapeutic target for this, so far, incurable disease. Previously, we reported on the role of the protein kinase-B/Akt in CVB3 replication and virus-induced cell injury. Here, we report on regulation of virus-induced Akt activation by the integrin-linked kinase in infected mouse cardiomyocytes and HeLa cells. This study also presents the first observation that inhibition of ILK in CVB3-infected cells significantly improves the viability of infected cells, while blocking viral replication and virus release. Complementary experiments using a constitutively active form of Akt1 revealed that the observed protective effect of ILK inhibition is dependent on the associated downregulation of virus-induced Akt activation. To our knowledge, this is the first report of such beneficial effects of ILK inhibition in a viral infection model and conveys new insights in our efforts to characterize a novel therapeutic target for treatment of enteroviral myocarditis.

Innate defense mechanism against virus infection within the cardiac myocyte requiring gp130-STAT3 signaling.

BACKGROUND: Little is known about innate immune mechanisms within the cardiac myocyte that determine susceptibility to enterovirus infection, an important cause of myocarditis and subsequent heart failure. Although interferon (IFN) generally plays a key role in innate immunity, ablation of IFN receptors has little or no effect on acute coxsackievirus B3 infection in the heart. Interestingly, gp130-cytokine-mediated stimulation of neonatal ventricular myocytes has a cytoprotective effect against virus infection in culture that can be inhibited by suppressors of cytokine signaling (SOCS)-3, a physiological inhibitor of gp130 signaling that does not affect IFN signaling. Therefore, we hypothesized that inhibition of gp130 signaling by SOCS3 would change cardiac myocyte susceptibility to virus infection without affecting IFN signaling. METHODS AND RESULTS: We generated cardiac-specific SOCS3 transgenic mice. Despite an intact IFN-mediated antiviral response in adult transgenic myocytes, there was a marked increase in susceptibility to viral infection in the SOCS3 transgenic mouse hearts. This indicated the presence of IFN-independent innate defense mechanisms within the cardiac myocyte. Subsequently, we demonstrated that cardiac-specific gp130-knockout mice also had increased susceptibility to viral infection. Furthermore, we demonstrated that the gp130-mediated increase in survival of infected myocytes occurred through a signal transducers and activators of transcription-3-dependent mechanism that did not affect viral replication. This was accompanied by a persistent expression of full-length dystrophin after coxsackievirus B3 infection. In addition, we found that both SOCS3 transgenic and gp130-deficient mice had a decrease in alpha-sarcoglycan. CONCLUSIONS: SOCS3-mediated regulation of gp130 signaling can affect susceptibility to viral infection in the heart. Increased cardiac cell survival through gp130-signal transducers and activators of transcription-3 signaling appears to play an important role in preserving nondividing cardiac myocytes until specific immune responses begin to clear the virus.

Defect of rabies virus phosphoprotein in its interferon-antagonist activity negatively affects viral replication in muscle cells.

Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-ß promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-ß-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.

Oxymatrine provides protection against Coxsackievirus B3-induced myocarditis in BALB/c mice.

Oxymatrine is the primary pharmacological component of Sophora flavescens Ait. In the present study, we investigated the protective effect of oxymatrine against Coxsackievirus B3-induced myocarditis in mice. Coxsackievirus B3-infected HeLa cells were treated with oxymatrine and the viral titer, as well as the degree of cellular proliferation were determined. Additionally, BALB/c mice were infected with Coxsackievirus B3 and received differing concentrations of oxymatrine. On days 5 and 12 following treatment, mice were sacrificed, and serum lactate dehydrogenase, creatine kinase-MB isozyme, and tumor necrosis factor-α levels were quantified. The heart index and degree of myocardial tissue inflammation were also assessed. On day 5, the Coxsackievirus B3 TCID50 values of the heart tissue, and the expression of NTR, IFN-γ, and TNF-α genes in the myocardial tissue were measured. Our results showed that oxymatrine exhibits potent antiviral effects on Coxsackievirus B3 as 50% inhibition was achieved at a concentration as low as 0.238 mg/mL. Oxymatrine markedly reduced the viral titer and inhibited cardiac myocyte pathology exhibited in viral myocarditis. Furthermore, oxymatrine treatment reduced the expression of Coxsackievirus B3 NTR and mouse TNF-α genes compared to the controls. Therefore, our findings indicate that oxymatrine is a promising potent antiviral agent against Coxsackievirus B3-induced myocarditis.

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo.

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

Allosteric Regulation of Phosphatidylinositol 4-Kinase III Beta by an Antipicornavirus Compound MDL-860.

MDL-860 is a broad-spectrum antipicornavirus compound discovered in 1982 and one of the few promising candidates effective in in vivo virus infection. Despite the effectiveness, the target and the mechanism of action of MDL-860 remain unknown. Here, we have characterized antipoliovirus activity of MDL-860 and identified host phosphatidylinositol-4 kinase III beta (PI4KB) as the target. MDL-860 treatment caused covalent modification and irreversible inactivation of PI4KB. A cysteine residue at amino acid 646 of PI4KB, which locates at the bottom of a surface pocket apart from the active site, was identified as the target site of MDL-860. This work reveals the mechanism of action of this class of PI4KB inhibitors and offers insights into novel allosteric regulation of PI4KB activity.

Enhancement of apoptosis via an extrinsic factor, TNF-alpha, in cells infected with cytopathic bovine viral diarrhea virus.

Isolates of bovine viral diarrhea virus (BVDV) are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. Calves persistently infected with ncp BVDV are known to develop lethal mucosal disease (MD) after superinfection by cp BVDV. Although the UV-irradiated supernatant of cp BVDV-infected cells has been reported to have no capacity to induce cell death, we found that it could enhance cell death through apoptosis. Up-regulation of tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) mRNAs was detected specifically in cp BVDV-infected primary cell cultures. Suppression of TNF-alpha via antisense oligonucleotide transfection or incubation with a polyclonal antibody against TNF-alpha resulted in attenuation of apoptosis induced by cp BVDV, suggesting that TNF-alpha participates in apoptosis execution. Although TNF-alpha is one of the iNOS-inducible factors, the iNOS up-regulation was not regulated by TNF-alpha. And iNOS was revealed to serve as anti-apoptotic factor, contrary to our expectation. In addition, the expression level of both TNF-alpha and iNOS mRNAs in the ncp BVDV-infected cells was kept lower than that in the mock-infected cells, suggesting that ncp BVDV reduced or interfered with the factor triggering the expression of both mRNAs. These characteristic mRNA transcriptions would help to explain why BVDV acts differently in cells as well as in vivo, depending on its biotype. To elucidate viral factors inducing TNF-alpha and iNOS may be critical to understand the mechanism of MD development, which closely correlates with cp BVDV-induced apoptosis.

RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies.

Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.