神经胶质瘤细胞ATF5表达水平对HCMV复制的影响

人巨细胞病毒(human cytomegalovirus,HCMV)在神经胶质瘤细胞中的复制水平不一,其机制尚不清楚。本研究通过下调转录激活因子5(ATF5)在神经胶质瘤细胞中的表达,检测HCMV感染神经胶质瘤细胞后病毒复制水平的变化。首先用HCMV AD169(MOI=5)分别感染U87、SY5Y及A172细胞,观察细胞形态变化,分别在24、48、72、96、120 h取各时间点上清液检测病毒滴度;Real-time PCR检测HCMV即刻早期基因(IE2)、早期基因(UL44)、晚期基因(UL99)及ATF5的表达情况;Western-blot检测病毒基因编码蛋白及ATF5表达的情况。结果显示HCMV在U87、SY5Y细胞中复制水平与病毒在A172细胞中复制水平相比,U87、SY5Y细胞组明显高于A172细胞组(P0.05),ATF5表达在U87、SY5Y细胞组与A172细胞组相比,U87、SY5Y细胞组ATF5表达明显高于A172组(P0.05);利用慢病毒介导的RNA干扰技术下调ATF5在U87、SY5Y细胞的表达,用HCMV感染细胞检测病毒基因及蛋白的表达,结果ATF5表达下调可抑制HCMV的复制(P0.05)。以上结果表明,在胶质瘤细胞中下调ATF5表达水平可以抑制HCMV的复制水平。     

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

PTMs such as phosphorylations are usually involved in signal transduction pathways. To investigate the temporal dynamics of phosphoproteome changes upon viral infection, a model system of IMR‐90 cells infected with human adenovirus type 2 (Ad2) is used in a time‐course quantitative analysis combining titanium dioxide (TiO₂) particle enrichment and SILAC‐MS. Quantitative data from 1552 phosphorylated sites clustered the highly altered phosphorylated sites to the signaling by rho family GTPases, the actin cytoskeleton signaling, and the cAMP‐dependent protein kinase A signaling pathways. Their activation is especially pronounced at early time post‐infection. Changes of several phosphorylated sites involved in the glycolysis pathway, related to the activation of the Warburg effect, point at virus‐induced energy production. For Ad2 proteins, 32 novel phosphorylation sites are identified and as many as 52 phosphorylated sites on 17 different Ad2 proteins are quantified, most of them at late time post‐infection. Kinase predictions highlighted activation of PKA, CDK1/2, MAPK, and CKII. Overlaps of kinase motif sequences for viral and human proteins are observed, stressing the importance of phosphorylation during Ad2 infection.     

Effect of 25-hydroxyvitamin D3 on rotavirus replication and gene expressions of RIG-I signalling molecule in porcine rotavirus–infected IPEC-J2 cells

The study evaluated whether a 25-hydroxyvitamin D₃ (25D₃) supplementation decreases the replication of rotavirus by the retinoic acid-inducible gene I (RIG-I) signalling pathway in a porcine small intestinal epithelial cell line (IPEC-J2). The results show that IPEC-J2 cells express high baseline levels of 1α-hydroxylase (CYP27B1), which converts inactive 25D₃ to the active 1,25-dihydroxyvitamin D₃ (1,25D₃). Porcine rotavirus (PRV) infection alone resulted in a significant increase in CYP27B1 mRNA, which augmented the production of active vitamin D. Physiological concentrations of 25D₃ were found to decrease PRV replication in IPEC-J2 cells. RIG-I plays an important role in the recognition of double-stranded RNA virus by host cells. Upon recognition, RIG-I triggers a series of signalling molecules such as interferon-β (IFN-β) promoter stimulator 1 (IPS-1) leading to the expression of type I interferons (IFN-β). Active 25D₃ that was generated by PRV-infected IPEC-J2 cells led to an increased expression of toll-like receptors 3 (TLR3), RIG-I, IPS-1, IFN-β and IFN-stimulated genes 15 (ISG₁₅) with important innate immune functions. Inhibiting CYP27B1 also failed to increase RIG-I, IPS-1, IFN-β and ISG₁₅ mRNA expression. These observations suggest that 25D₃ can directly inhibit PRV in IPEC-J2 cells, which requires this active form of vitamin D. The anti-rotavirus effect of 25D₃ is mediated at least in part by RIG-I signalling pathways in IPEC-J2 cells.     

Pseudorabies virus US3 protein kinase mediates actin stress fiber breakdown

Disruption of specific components of the host cytoskeleton has been reported for several viruses and is thought to be beneficial for viral replication and spread. Our previous work demonstrated that infection of swine kidney (SK-6) cells with pseudorabies virus (PRV), a swine alphaherpesvirus, induced actin stress fiber breakdown. In the present study, using several PRV deletion mutants, we found that the US3 serine/threonine (S/T) protein kinase is involved in breakdown of actin stress fibers in different PRV-infected cell lines. Further, by transfection assays, we showed that PRV US3 itself, in the absence of other viral proteins, is able to trigger actin stress fiber breakdown when it is localized in sufficient amounts in the nucleus.     

The nonstructural protein 2C of Coxsackie B virus has RNA helicase and chaperoning activities

RNA-remodeling proteins, including RNA helicases and chaperones, play vital roles in the remodeling of structured RNAs. During viral replication, viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements. Coxsackieviruses B3 (CVB3) and Coxsackieviruses B5 (CVB5), belonging to the genus Enterovirus in the family Picornaviridae, have been reported to cause various infectious diseases such as hand-foot-and-mouth disease, aseptic meningitis, and viral myocarditis. However, little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins. In this study, we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A, B, and C motifs, and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner, but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP. In addition, we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions. Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.     

Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infection affect the phosphorylation of MAPK and Bcl-2 and these proteins may be the common element linking RIP and viral infection. This study investigated the effect of HSV-1 infection on p38 MAPK and Bcl-2 as well as possible interference by TCS. Results showed that HSV-1 infection induced an elevation of phosphorylated p38 and Bcl-2 in Vero cells, which could be partially blocked by TCS. At the same time, both viral replication and host cells viability were lowered. Viral replication, Vero cell viability, p38 MAPK and Bcl-2 were further reduced with the addition of a p38 MAPK inhibitor (SB203580). This suggested that TCS may interfere with MAPK and Bcl-2 signals generated by infection leading to inhibition of viral replication. In summary, our results demonstrated that HSV-1 infection in Vero cells induced an elevation of p38 MAPK and Bcl-2. TCS suppressed this rise and reduced viral replication. The MAPK family may play a role in the antiviral mechanism of TCS.     

Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine

Pseudorabies virus (PRV) is a herpesvirus of swine, a member of the Alphaherpesvirinae subfamily, and the etiological agent of Aujeszky's disease. This review describes the contributions of PRV research to herpesvirus biology, neurobiology, and viral pathogenesis by focusing on (i) the molecular biology of PRV, (ii) model systems to study PRV pathogenesis and neurovirulence, (iii) PRV transsynaptic tracing of neuronal circuits, and (iv) veterinary aspects of pseudorabies disease. The structure of the enveloped infectious particle, the content of the viral DNA genome, and a step-by-step overview of the viral replication cycle are presented. PRV infection is initiated by binding to cellular receptors to allow penetration into the cell. After reaching the nucleus, the viral genome directs a regulated gene expression cascade that culminates with viral DNA replication and production of new virion constituents. Finally, progeny virions self-assemble and exit the host cells. Animal models and neuronal culture systems developed for the study of PRV pathogenesis and neurovirulence are discussed. PRV serves as a self-perpetuating transsynaptic tracer of neuronal circuitry, and we detail the original studies of PRV circuitry mapping, the biology underlying this application, and the development of the next generation of tracer viruses. The basic veterinary aspects of pseudorabies management and disease in swine are discussed. PRV infection progresses from acute infection of the respiratory epithelium to latent infection in the peripheral nervous system. Sporadic reactivation from latency can transmit PRV to new hosts. The successful management of PRV disease has relied on vaccination, prevention, and testing.     

Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

Background

Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.

Principal Findings

We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.

Conclusions

Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.     

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

The NS5A protein of the hepatitis C virus (HCV) is an integral component of the viral replicase. It also modulates cellular signaling and perturbs host interferon responses. The multifunctional characteristics of NS5A are mostly attributed to its ability to interact with various cellular proteins. This study aimed to identify the novel cellular factors that interact with NS5A and decipher the significance of this interaction in viral replication. The NS5A-interacting proteins were purified by the tandem affinity purification (TAP) procedure from cells expressing NS5A and identified by mass spectrometry. The chaperone protein Hsp72 was identified herein. In vivo protein-protein interaction was verified by co-immunoprecipitation and an in situ proximity ligation assay. In addition to NS5A, Hsp72 was also associated with other members of the replicase complex, NS3 and NS5B, suggesting that it might be directly involved in the HCV replication complex. Hsp72 plays a positive regulatory role in HCV RNA replication by increasing levels of the replicase complex, which was attributed either to the increased stability of the viral proteins in the replicase complex or to the enhanced translational activity of the internal ribosome entry site of HCV. The fact that the host chaperone protein Hsp72 is involved in HCV RNA replication may represent a therapeutic target for controlling virus production.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

MEK5/ERK5/mef2: A novel signaling pathway affected by hepatitis C virus non-enveloped capsid-like particles

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.