DEAD-box RNA解旋酶家族在天然免疫应答信号通路中的作用

病毒入侵宿主细胞时,宿主细胞启动抑制病毒复制的免疫机制.同样,病毒也会利用多种手段去逃避先天免疫感应机制的监测以及宿主细胞对外来者的降解,同时还会操纵宿主细胞为自身的增殖提供便利.DEAD-box解旋酶家族是一类存在于宿主细胞中的功能蛋白,它们在转录、剪接、mRNA的合成和翻译等多种细胞过程中起着关键作用.该家族成员拥...     

Fiber-modified adenovirus vectors decrease liver toxicity through reduced IL-6 production

Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c(+) cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses.     

口蹄疫病毒与宿主细胞的相互作用研究进展

口蹄疫病毒（FMDV）导致了偶蹄动物口蹄疫的发生,它是一类有着自身特点的RNA病毒。首先,FMDV衣壳蛋白VP1识别结合宿主细胞膜上的整联蛋白等受体,以内吞的方式进入细胞,利用宿主细胞成分完成病毒蛋白的合成。这些新合成的L^pro、2C和3C^pro等病毒致病因子进一步抑制宿主基因的转录和翻译,诱导细胞凋亡和白噬,并抑制干扰素介导的一系列先天性和获得性免疫反应。宿主则在病毒侵染细胞的初期,利用病毒识别受体等来识别病毒并诱导合成干扰素等细胞因子,介导多种免疫反应以清除病毒。病毒和宿主两者在持续的利用和较量中完成疾病的发生和痊愈等。其次,不断发现的病毒受体、结合基序、致病因子及宿主细胞的多种免疫调节因子将成为相关领域新的研究内容。综上,开发高效安全疫苗、增强自身免疫力及利用RNAi直接抑制病毒RNA等便成为现代FMDV防治的主要内容。     

A baculovirus-encoded MicroRNA (miRNA) suppresses its host miRNA biogenesis by regulating the exportin-5 cofactor Ran

MicroRNAs have emerged as key players in the regulation of various biological processes in eukaryotes, including host-pathogen interactions. Recent studies suggest that viruses encode miRNAs to manipulate their host gene expression to ensure their effective proliferation, whereas the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we demonstrate that an insect virus, Bombyx mori nucleopolyhedrosis virus (BmNPV), modulates the small-RNA-mediated defense of its host, B. mori, by encoding an miRNA (bmnpv-miR-1) that downregulates the expression of the host GTP-binding nuclear protein Ran, an essential component of the exportin-5-mediated nucleocytoplasmic transport machinery mainly involved in small-RNA transport from the nucleus to the cytoplasm. We demonstrate the sequence-dependent interaction of bmnpv-miR-1 with Ran mRNA using cell culture and in vivo assays, including RNA interference (RNAi) of Ran. Our results clearly show that bmnpv-miR-1 represses Ran, leading to reduction in the host small-RNA population, and consequently, the BmNPV load increases in the infected larvae. Blocking of bmnpv-miR-1 resulted in higher expression levels of Ran and a decrease in BmNPV proliferation. In contrast, blockage of host miRNA, bmo-miR-8, which targets the immediate-early gene of the virus and whose production was repressed upon bmnpv-miR-1 and Ran dsRNA administration, resulted in a significant increase in the virus load in the infected B. mori larvae. The present study provides an insight into one of the evasion strategies used by the virus to counter the host defense for its effective proliferation and has relevance to the development of insect virus control strategies.     

Adenoviral expression of suppressor of cytokine signaling-1 reduces adenovirus vector-induced innate immune responses

Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells.

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.     

Selective translation of mengovirus RNA over Host mRNA in homologous, fractionated, cell-free translational systems from Ehrlich-ascites-tumor cells.

The selective translation of viral RNA in mengovirus-infected Ehrlich ascites tumor cells was investigated using fractionated translational systems whose macromolecular components were derived entirely from uninfected or virus-infected cells. Both systems translate host mRNA from uninfected cells, host mRNA from virus-infected cells, and mengovirus RNA. In competition experiments, where viral RNA and host mRNA were translated together in systems from uninfected cells, the relative amounts of virus-specific and host-specific proteins synthesized were proportional to the relative concentrations of the RNA templates. In systems whose components were obtained from virus-infected cells, mengovirus RNA was preferentially translated. 70% of the selectivity found in the translational systems derived from infected cells was due to the initiation factor fraction, the remaining 30% to components of the pH 5 enzyme fraction. In addition, host mRNA isolated after virus infection is translated in vitro to a lower extent in the presence of mengovirus RNA than is host mRNA from uninfected cells.     

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.     

Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.     

Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.