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.     

Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile viral gastroenteritis and are responsible for up to 700,000 deaths each year (6, 24). The RV genome consists of 11 segments of double-stranded RNA (dsRNA) which can be separated by polyacrylamide gel electrophoresis (PAGE), resulting in typical dsRNA profiles exhibiting four well-defined size classes of segments. However, some RV show unusual dsRNA profiles in which standard-sized segments are replaced by larger, rearranged forms (for a review, see reference 5). Gene rearrangements were first reported for RV isolated from immunodeficient children with chronic infection (11, 26) and can be obtained experimentally by serial passages at a high multiplicity of infection (MOI) in cell culture (10, 14, 21). We recently showed that rearrangements also occur during acute RV infection (36). Gene rearrangement usually consists of a partial head-to-tail duplication of a segment sequence. In most cases, sequence duplication occurs after the stop codon, leaving the open reading frame (ORF) untouched and the encoded protein unchanged (5, 6). Less frequently, the duplication occurs within the ORF, which thus encodes a modified protein (8, 38).Manipulation at the cDNA level of most positive- and negative-strand RNA viral genomes, followed by rescue of infectious virus, is now well established and has provided a better understanding of RNA virus replication and pathogenesis. In the case of Reoviridae, the development of reverse genetics systems has been hampered by the nature of the genome, which carries 10 to 12 dsRNA segments that are densely packed within the viral particle and are transcribed and replicated within a subviral structure. Recent major advances were obtained in the development of reverse genetics systems for some Reoviridae. For mammalian orthoreo- and orbiviruses, reverse genetics systems based on the transfection of plasmid cDNAs (13) or cDNA-derived mRNAs (2) corresponding to a complete set of 10 RNA segments have been established, allowing the rescue of infectious viral progeny. However, for RV, there is no report indicating that transfection of a complete set of viral mRNAs can result in the rescue of infectious viruses, and it is still unknown whether the RV genome can be infectious. In 2006, Komoto et al. (16) described the first reverse genetics system for RV, based on a model originally developed for influenza viruses by Palese and colleagues (17). In this system, an exogenous RV mRNA synthesized in the cell cytoplasm by the T7 RNA polymerase (T7pol), expressed by a recombinant vaccinia virus, is packaged in place of its homologous counterpart into a helper RV. This reverse genetics system has allowed the recovery of engineered monoreassortant infectious RV (designated recombinant RV) with an incorporated exogenous segment 4 encoding the spike protein VP4. An in vitro-modified cDNA-derived segment 4 was also successfully introduced into a recombinant RV to obtain a virus carrying a VP4 antigenic chimera (15). However, this system is restricted to segments encoding antigenically distinct viral surface proteins (like VP4) because the selection of recombinant viruses requires the use of specific and potent neutralizing antibodies to eliminate wild-type (WT) helper viruses.Results obtained from a preliminary study suggested that rearranged RNA segments might overcome this limitation. Indeed, we first characterized human RV clones containing rearranged segment 7, 11, or both (8) and then compared the fitness levels of rearranged versus WT viruses. We found that viruses with rearranged segment 7 or 11 replicated less well than or equally to WT viruses, as judged by viral growth curve experiments. Surprisingly, in competition growth experiments, rearranged segment 7 or 11 was always selected into the viral progenies, even when mixed infections were performed with a ratio of 1 rearranged to 1,000 WT viruses (4). The absence of a growth advantage conferred on the virus by rearranged segments, combined with their preferential segregation into the viral progenies, suggested that rearranged RNA segments might be packaged preferentially. These observations are in agreement with results of earlier studies showing that rearranged segment 5 or 11 segregated preferentially into viral progenies issued from mixed infections with WT virus (10, 19).We developed a reverse genetics system for RV on the basis of the preferential packaging of rearranged RNAs. We report here the rescue of recombinant viruses carrying cDNA-derived rearranged segment 7 (either unmodified or containing silent mutations introduced by site-directed mutagenesis to generate restriction enzyme sites as markers), with no selection pressure other than serial passage in cell culture. We also report the rescue of a recombinant virus expressing a double-sized recombinant NSP3 protein encoded by an in vitro-modified cDNA-derived rearranged segment 7, showing for the first time that an in vitro-engineered gene encoding a modified nonstructural protein can be introduced into an infectious RV.     

Structural insights into small RNA sorting and mRNA target binding by Arabidopsis Argonaute Mid domains

The RISC-associated Argonaute (Ago) proteins play the catalytic role for RISC-mediated gene regulation by selecting small RNAs and subsequent targeting and cleavage of complementary mRNAs. Ago Mid domains are proposed to play essential roles in small RNA sorting. Here, we report the crystal structures of Arabidopsis Ago1 Mid domain and its chimera mutant with part of Ago1 replaced by Ago4. The structures demonstrate that a single amino insertion in the nucleotide specificity loop of AtAgo1 will change the nucleotide binding preference of AtAgo1 from “5′-U” to “5′-A”. Moreover, we identify a long positively charged groove located along the “5′-end-nucleotide specificity loop” and occupied by several sulfate ions with the distance of 9-11 Å distance, indicating a putative mRNA target binding groove.     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

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.     

Regulation of Human Immunodeficiency Virus Replication by 2′,5′-Oligoadenylate-Dependent RNase L

Activation of RNase L by 2′,5′-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1 nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.     

Le « nucléus Donggutuo » et sa signification dans l’industrie du paléolithique inférieur de Donggutuo, bassin de Nihewan, Chine du Nord

As one of the most important Lower Pleistocene sites of the Nihewan basin in North China, Donggutuo (DGT) site is well known for its fine retouched small tools and characters of flake industry. However, new excavations still reveal some new discoveries and educe some new issues to us. For example, the “DGT core” introduced in this article is a new discovered typotechnology and indicates new economic strategy of the local people, which was never known before in this area at the time of 1.1 Ma B.P. The paper provides general background and statistic analysis of DGT industry including special method applied to the “DGT core” and discusses possible influence of environmental change for the emerging of the “DGT core”. The “DGT core” shows a germinated microlithic tradition and reflects a cultural diversity of early humans in the Lower Paleolithic in North China.     

Antiviral ribozymes

Catalytic RNAs are a genetic property not only of some particular viroids or viruses, but also are more common naturally among eukaryotes and even prokaryotes than earlier expected. However, the major interest in ribozymes results from their potential for development of “tailor-made” cDNA constructions designed to be transcribed into catalytic RNAs that will recognize by hybridization and destroy by specific cleavage their cellular or viral RNA targets. The efficiency of an antiviral ribozyme is determined by both the accessibility and sequence conservation of the target region, as well as the design of the ribozyme: its type, size, and composition of flanking sequences; expression rates; and cellular compartment localization. Until now the most frequently selected viral target is the human immunodeficiency virus, where an up to a 10⁴-fold inhibition in its progeny production has been achieved. Although the first generation ribozymes focused on improvements in basic design and expression rates, more recently the efficiency of antiviral catalytic activity has been increased by employing polyribozymes and/or multitarget ribozymes, as well as special constructions to enhance the cellular co-compartmentation of the ribozyme with its viral RNA target.     

Fluorescence cross-correlation spectroscopy reveals mechanistic insights into the effect of 2'-O-methyl modified siRNAs in living cells

RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of 2′-O-methyl (2′OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2′OMe nucleotides on the 3′-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand asymmetry, or target recognition. 2′OMe modifications introduced at the 5′-end reduced activated RISC stability, whereas incorporations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand. Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo properties.     

Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses

Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals – herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids.