MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including Thogoto virus (THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.     

Rescue of influenza C virus from recombinant DNA

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3' and 5' noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10(3) and 10(4) PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.     

Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.     

Rescue of Influenza A Virus from Recombinant DNA

We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.     

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.     

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.     

重组新城疫病毒Anhinga株与TRAIL蛋白协同杀伤肿瘤细胞

将新城疫病毒（Newscastle disease virus, NDV）Anhinga株的全 长基因组cDNA克隆质粒、pTM1-L、pTM1-P、pTM1-NP表达质粒共转染稳定表 达T7 RNA聚合酶的BSRT7/5细胞,得到拯救NDV病毒．通过PCR、酶切法、测 序证明拯救病毒中存在引入的分子标签.通过血凝实验、蚀斑测定证明成功 拯救病毒.并研究了该重组病毒对4种不同人肿瘤细胞的体外杀伤效果．首 次证明重组Anhinga株对SMMC-7721细胞、A549细胞、HepG2细胞和SH-SY5Y 细胞均有杀伤作用．该重组病毒主要诱导SMMC-7721细胞和A549细胞凋亡, 诱导HepG2细胞和SH-SY5Y细胞坏死．TNF家族成员TRAIL蛋白可以显著增强 NDV杀伤肿瘤细胞的效果.本实验为进一步研究重组NDV用于肿瘤治疗奠定基 础．     

Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.     

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.     

Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids.

We have shown previously that COS-1 cells infected with a vaccinia virus recombinant (vTF7-3) which expresses the T7 RNA polymerase gene and then transfected with four pGEM-derived plasmids encoding the influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA polypeptides) can express a synthetic influenza virus-like chloramphenicol [correction of chloramphenical] acetyltransferase (CAT) RNA (I. Mena, S. de la Luna, C. Albo, J. Martín, A. Nieto, J. Ortín, and A. Portela, J. Gen. Virol. 75:2109-2114, 1994). Here we demonstrate that by supplying the vTF7-3-infected cells with plasmids containing cDNAs of all 10 influenza virus-encoded proteins, the transfected CAT RNA can be expressed and rescued into particles that are budded into the supernatant fluids. The released particles can transfer the enclosed CAT RNA to MDCK cultures and resemble true influenza virions in that they require trypsin treatment to deliver the RNA to fresh cells and are neutralized by a monoclonal antibody specific for the influenza A virus hemagglutinin. Moreover, analysis by electron microscopy showed that the culture medium harvested from the transfected cells contained vesicles that could be labeled with an anti-HA monoclonal antibody and that were similar in size and morphology to authentic influenza virus particles. It is also shown that detection of recombinant particles capable of transmitting the CAT RNA does not require expression of the influenza virus nonstructural protein NS1. All of these data indicate that influenza virus-like particles enclosing a synthetic virus-like RNA can be assembled in cells expressing all viral structural components from recombinant plasmids.