Introduction of site-specific mutations into the genome of influenza virus.

We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with defined biological properties.     

Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

The family Reoviridae is a nonenveloped virus group classified into 15 genera. These viruses have double-stranded (ds) RNA genomes with 9 to 12 segments. This family includes several important pathogens in both humans and animals. Mammalian orthoreovirus (MRV) and Nelson Bay orthoreovirus (NBV), which belong to the genus Orthoreovirus, have genomes consisting of 10 segments of dsRNA. MRV is an experimental model for studies of Reoviridae virus replication and pathogenesis. NBV, classified into the fusogenic subgroup of this genus, is associated with acute respiratory tract infections in humans (1–3). The fusogenic orthoreoviruses encode a unique fusion-associated small transmembrane (FAST) protein associated with cell–cell fusion and viral pathogenesis (4–6). Bluetongue virus (BTV) and African horse sickness virus (AHSV) belong to the genus Orbivirus, have genomes with 10 segments of dsRNA, and cause severe diseases in domestic animals (7, 8). Rotavirus (RV, genus Rotavirus) has a genome with 11 segments of dsRNA and causes severe diarrhea in young children. RV infection is responsible for 128,500 deaths per year worldwide, predominately in developing countries (9). Colorado tick fever virus (CTFV), an arthropod-borne virus transmitted by ticks and belonging to the genus Coltivirus, has a dsRNA genome comprising 12 segments. CTFV causes a variety of symptoms in humans, including abrupt fever, chills, headache, myalgia, and abdominal pain (10, 11). Within the genus Coltivirus (in addition to CTFV), Eyach virus, Shelly Headland virus, Kundal virus, and Tarumizu tick virus (TarTV) have been isolated from, or detected in, ticks in Europe, Australia, India, and Japan, respectively (12–17). Taï Forest reovirus was isolated from free-tailed bats in Côte d’Ivoire (18), and Lishui pangolin virus was detected in pangolins in China (19). These reports suggest that coltiviruses are distributed in multiple species worldwide. However, the molecular mechanism underlying the propagation and pathogenesis of these viruses remains largely unknown.Reverse genetics systems are powerful tools used to study many aspects of viral biology and virus–host interactions and also provide an opportunity to generate recombinant viruses that can be used for vaccines or as viral vectors. This technology has been used for several viruses in the family Reoviridae. In the genus Orthoreovirus, an entirely plasmid-based reverse genetics system was developed for MRV in 2007 (20). This is the first example of engineering recombinant Reoviridae viruses entirely from cloned complementary DNAs (cDNAs). This system was established by cotransfection of cloned cDNAs representing 10 MRV gene segments, each flanked by the T7 promoter and hepatitis delta virus (HDV) ribozyme, into cells expressing T7 RNA polymerase. Subsequently, a reverse genetics system for NBV, belonging to a fusogenic reovirus group, was established based on the MRV rescue system (21). In the genus Orbivirus, RNA- and DNA-based reverse genetics systems were developed for BTV, AHSV, and Epizootic hemorrhagic disease virus (22–25). Although development of a reverse genetics system for the genus Rotavirus has lagged behind those for the genera Orthoreovirus and Orbivirus, our group recently developed the first plasmid-based reverse genetics system for RV by cotransfecting plasmids encoding unique heterogeneous viral proteins, NBV FAST, and vaccinia virus capping enzyme as rescue enhancers along with the 11 RV T7 promoter-based rescue plasmids (26). In addition, reverse genetics systems were also developed for Bombyx mori cypovirus and Dendrolimus punctatus cypovirus (genus Cypovirus), which are insect pathogens and have a genome consisting of 10 segments of dsRNA (27, 28). Since reverse genetics systems were developed for Reoviridae viruses with genomes containing 10 or 11 segments of dsRNA, studies of the family Reoviridae have advanced markedly. However, to date, no reverse genetics system has been established for Reoviridae viruses with 12 genome segments.In this study, we isolated a TarTV strain from a raccoon dog postmortem. Using this TarTV strain, we established an entire plasmid-based reverse genetics system and rescued VP12 mutant viruses and a HiBiT-tagged reporter virus. This system is a useful tool to generate recombinant coltiviruses with 12 dsRNA genome segments.     

Method for introducing site-specific mutations into adenovirus 2 genome: construction of a small deletion mutant in VA-RNAI gene.

We have developed a method for introducing mutations into the adenovirus type 2 genome at predetermined sites. Specific mutations are introduced into segments of the viral genome cloned in bacteria by using plasmid vectors. The chimeric DNA is used to construct viral mutants by cotransfection with two viral DNA segments derived from both ends of the viral genome, each of which has overlapping sequence homology with the cloned viral DNA segment. To illustrate this procedure, we cloned a large restriction fragment [EcoRI fragment A, map position (mp) 0--58.5] by using plasmid vector pBR322, and a small deletion mutation was introduced at the BamHI cleavage site located within the VA-RNAI gene (mp 29 at residue +75 on VA-RNAI gene). The mutated DNA was then used to construct viral mutants by cotransfection into human cells with the adenovirus type 2 DNA--protein complex digested with Sal I. In vivo recombination occurred via overlapping sequences between the cloned EcoRI fragment A and Sal I-digested DNA--protein complex at two sites, mp 0--25 (i.e., within Sal I fragment B) and mp 45--58.5 (i.e., overlapping sequences between Sal I fragment A and EcoRI fragment A), generating infectious DNA molecules with intact ends. The viral mutant grows as well as the wild type in KB cells and induces the synthesis of smaller VA-RNAI but normal-size VA-RNAII.     

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoV_U201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient’s stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.Human noroviruses (HuNoVs) belong to the genus Norovirus of the family Caliciviridae and are the predominant cause of epidemic and sporadic cases of acute gastroenteritis worldwide (1, 2). HuNoVs are spread through contaminated water or food, such as oysters, shellfish, or ice, and by person-to-person transmission (3, 4). Although HuNoVs were identified more than 40 y ago, our understanding of the replication cycle and mechanisms of pathogenicity is limited, because these viruses remain noncultivatable in vitro, a robust small animal model to study viral infection is not available, and reports of successful passage of HuNoVs in a 3D cell culture system have not been reproduced (5–7). Recently, a murine model for HuNoV infection was described that involves intraperitoneal inoculation of immunocompromised mice (8); its generalizability and robustness for studying individual HuNoVs and many aspects of HuNoV biology remain to be established. Gnotobiotic pigs can support replication of a HuNoV genogroup II (GII) strain with the occurrence of mild diarrhea, fecal virus shedding, and immunofluorescent (IF) detection of both structural and nonstructural proteins in enterocytes (9). Previous systems to express the HuNoV genome from cloned DNA using T7/vaccinia systems showed that mammalian cells can produce progeny virus (10, 11), but these systems are not sufficiently efficient to be widely used to propagate HuNoVs in vitro. The factors responsible for the block(s) of viral replication using standard cell culture systems remain unknown.The HuNoV genome is a positive-sense ssRNA of ∼7.6 kb that is organized in three ORFs: ORF1 encodes a nonstructural polyprotein, and ORF2 and ORF3 encode the major and minor capsid proteins VP1 and VP2, respectively. Because of the lack of an in vitro system to propagate HuNoV, features of their life cycle have been inferred from studies using other animal caliciviruses and murine NoV (MNV) that can be cultivated in mammalian cell cultures (12). A 3′ coterminal polyadenylated subgenomic RNA is produced within infected cells. Both genomic and subgenomic RNAs have the same nucleotide sequence motif at their 5′ ends, and they are believed for HuNoVs and shown for MNV to be covalently linked to the nonstructural protein VPg at the 5′ ends (10, 13). During MNV infection of cells, nonstructural proteins are expressed from genomic RNA and form an RNA replication complex that generates new genomic RNA molecules as well as subgenomic RNAs encoding VP1, VP2, and the unique protein called VF1 (14). After expression of the structural proteins from subgenomic RNA molecules, the capsid is assembled, and viral RNA is encapsidated before progeny release. Previous reverse genetics systems for HuNoV used helper vaccinia MVA/T7 virus-based systems. Although helper virus-free systems have been developed for MNV (15, 16), no such system is available for HuNoVs. To overcome these problems, we established a reverse genetics system driven by a mammalian elongation factor-1α (EF-1α) promoter without helper virus and then modified this system to package a reporter gene (GFP) into ORF1.     

Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens

Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.     

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.     

Localization of genes for the double-stranded RNA killer virus of yeast.

The M double-stranded RNA (ds RNA) genome segment of the cytoplasmically inherited killer virus of yeast codes for two polypeptides when denatured and translated in vitro: a previously known 32,000-dalton peptide and a newly discovered 19,000-dalton peptide (NaDodSO4/polyacrylamide gel electrophoresis). An internal 190-base-pair region of the ds RNA is selectively degraded by S1 nuclease treatment at 65 degrees C, resulting in two ds RNA fragments which contain the termini of the original ds RNA. The larger fragment codes for the 32,000-dalton polypeptide and the smaller fragment codes for the 19,000-dalton polypeptide. Thus, the two gene products of M are encoded by distinct regions of this ds RNA.     

Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae.

Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcm1 mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak- phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to pet17. The mak- mutations in the trichodermin-resistant strains did not complement mak8-1, indicating that MAK8 and TCM1 are the same gene. The mak8-1 mutation does not make strains resistant to trichodermin, and one tcm1 mutation is only slightly mak-. Whereas tcm1 mutants lose M1 or M2 ds RNA, they do not lose L ds RNA. Because TCM1 codes for ribosomal protein L3 [Fried, H. M. & Warner, J. R. (1981) Proc. Natl. Acad. Sci, USA 78, 238--242], we conclude that ribosomal protein L3 is involved in the replication and maintenance of M ds RNA. Mutations in cyh2 or cry1, producing resistance to cycloheximide and crytopleurine due to mutant ribosomal proteins, do not produce a mak- phenotype. In analogy with bacterial ribosome assembly mutants, yeast low-temperature-sensitive (lts) mutants may have defective ribosomes. We thus examined mutants for an effect on the killer system. An lts5 mutant, unable to grow at 5 degrees C, also has a mak- phenotype (at 30 degrees C) that cosegregates in meiosis with the lts- phenotype. Mutations in seven other lts genes do not result in the mak- phenotype.     

In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus.

A system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments. The resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the phi 6 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.     

Gene targeting in the mouse: advances in introduction of transgenes into the genome by homologous recombination

The ability to stably introduce genes into the germline of animals provides a powerful means to address the genetic basis of physiology. Introduction of genes to generate transgenic animals has facilitated the development of complex genetic models of disease, as well as the in vivo study of gene function. However, one drawback of traditional transgenic technologies in which genes are microinjected into early-stage embryos is that there is little control over where and in how many copies genes are introduced into the genome. The development of animal transgenic technologies, which take advantage of homologous recombination mechanisms and the manipulation of embryonic stem (ES) cells, allows investigators to target and alter specific loci. In mouse transgenic systems, a plethora of sophisticated gene-targeting strategies now permit investigators to manipulate the genome in ways that essentially allow one to introduce virtually any desired change into the genome. Fur-thermore, when coupled with systems that allow for conditional gene expression, these gene-targeting strategies allow both temporal and tissue specific control of alterations to the genome. In the present review we briefly discuss some of the more recent gene-targeting strategies that have been developed to address the limitations of traditional animal transgenesis.     

Reverse dot-blot detection of the African-American beta-thalassemia mutations

DNA-based diagnosis of the beta thalassemias provides accuracy to newborn screening genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different-beta-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the beta-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two beta-globin target DNA fragments that encompass all known African-American beta-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele- specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 beta- thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common beta-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common beta-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of beta thalassemia among African-Americans and other ethnic groups in which beta thalassemia occurs.