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PubMed1997

Genome nucleotide lengths that are divisible by six are not essential but enhance replication of defective interfering RNAs of the paramyxovirus simian virus 5.

For some members of the Paramyxoviridae family of negative strand RNA viruses, efficient genome replication only occurs when the total genome length is a multiple of six (6N length, where N is any integer). To determine if this "rule of six" requirement applied to the replication of the prototype paramyxovirus simian virus 5 (SV5), defective interfering (DI) RNA genomes were generated by sequential undiluted passage of virus in tissue culture. Molecular cloning and nucleotide sequence analysis of 10 RNA genomes revealed a series of copyback DI RNAs with chain lengths between 449 and 1365 bases, but only 4 of the 10 naturally occurring RNA genomes were of 6N length. Many of the cloned DI genomes could be grouped into two distinct nested sets, with the members of each set having the same polymerase crossover junctions and extent of terminal complementarity but differing from each other by internal deletions. One of these nested sets of genomes consisted of novel DI RNAs that contained a pentameric stretch of nontemplated adenosine residues inserted precisely at the polymerase crossover junction. A reverse genetics system was established in which SV5 DI genomes were replicated in vivo entirely by cDNA-derived components. Using this system, two naturally occurring SV5 DI RNAs were examined in a mutational analysis to determine the role of genome length on SV5 RNA replication. The progressive insertion of one to six nucleotides into a 6N length DI genome (852 bases) resulted in a reduction in replication for RNAs that contained one to four additional bases (approximately 35-50% of WT levels), followed by an increase back to WT replication levels for genomes that were altered by five and six base insertions (approximately 70 and 100% of WT levels, respectively). An insertion of five nucleotides into a second non-6N length DI RNA (499 total bases) created a genome length that was a multiple of six (504 bases) and led to a approximately 10-fold stimulation of replication over that of the unaltered genome. Together, these results indicate that there was a clear influence of 6N genome length on SV5 DI RNA replication, but the stringency of this replication requirement appeared to be less than that found previously for other paramyxoviruses. This work completes the testing of the rule of six replication requirement for representatives of each of the four genera of the Paramyxoviridae family and indicates that the preference for replication of 6N length RNA genomes varies between the individual paramyxoviruses.

Animals↗
PubMed1998

Molecular basis for naturally occurring elevated readthrough transcription across the M-F junction of the paramyxovirus SV5.

Transcription of the paramyxovirus RNA genome is thought to involve a sequential stop-start mechanism whereby monocistronic mRNAs are produced by polyadenylation and termination of 3' upstream gene followed by reinitiation at the downstream start site. For a number of paramyxoviruses, transcription across the M-F gene junction results in the synthesis of high levels of a dicistronic M-F readthrough RNA. In cells infected with the paramyxovirus SV5, 15% or less of the transcripts from the viral P, M, SH, HN, and L genes were detected as readthrough products with the 3' proximal gene. By contrast, approximately 40% of the SV5 F mRNA was detected as a dicistronic M-F transcript. A comparison of the individual SV5 gene junctions showed that elevated M-F readthrough transcription correlate with the M gene end having the shortest U tract for directing polyadenylation and a gene end sequence that differs from the consensus sequence. We have tested the hypothesis that elevated M-F readthrough transcription results from an inefficient termination signal at the end of the M gene. A reverse genetics system was established whereby SV5 transcription was reconstituted in transfected cells using cDNA-derived polymerase components and dicistronic minigenomes that encoded either the SV5 M-F or the SH-HN gene junction. Chimeric SV5 minigenomes were constructed to contain exchanges of a 10 base gene end sequence and the U tract from the M-F (approximately 40% readthrough) and SH-HN (approximately 15% readthrough) junctions. Northern blot analysis of RNA synthesized from these altered templates showed that, in the context of the M-F intergenic region, increasing the length of the M gene end U tract from four residues to six or eight U residues did not decrease M-F readthrough transcription. In contrast, chimeric minigenomes that contained the 10 base region from the end of the SH gene directed very efficient gene termination and a corresponding decrease in readthrough transcription. Mutational analysis showed that a single G to A substitution located five bases 3' to the M gene U tract was sufficient to convert the M gene end region to an efficient signal for polyadenylation-termination. These results demonstrate a role for the gene end region located immediately 3' to the U tract as a major determinant of transcription termination in the paramyxovirus genome. The possible role of M-F readthrough transcription in the paramyxovirus growth cycle is discussed.

Animals↗
PubMed1998

The paramyxovirus SV5 small hydrophobic (SH) protein is not essential for virus growth in tissue culture cells.

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5DeltaSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5DeltaSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5DeltaSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5DeltaSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype.

Animals↗
PubMed2000

V and C proteins of measles virus function as virulence factors in vivo.

The measles virus (MV) P gene encodes three proteins: the P protein and two nonstructural proteins, C and V. Because the functions of both the C and V protein are unknown, we used MV C (C-) and V (V-) deletion recombinants generated by the MV reverse genetics system (F. Radecke, P. Spielhofer, H. Schnieder, K. Kaelin, M. Huber, C. Dotsch, G. Christiansen, and M. A. Billeter 1995. EMBO J. 14, 5773-5784). Compared to parental vaccine strain, Edmonston (Ed) MV, both had normal growth and cytopathic effects in Vero cells and showed similar growth kinetics in human neuroblastoma SK-N-MC cells and in primary mouse neurons expressing the MV receptor, CD46. However, in vivo, using YAC-CD46 transgenic mice as a model for MV induced CNS disease (M. B. A. Oldstone, H. Lewicki, D. Thomas, A. Tishon, S. Dales, J. Patterson, M. Manchester, D. Homann, D. Naniche, and A. Holz 1999. Cell 98, 629-640), C- and V- viruses differed markedly from wt Ed(V(+)C(+)) virus. Newborn mice inoculated with as little as 10(3) PFU of Ed strain became ill and died after 10-15 days. In contrast, those inoculated with 10(3) or 10(4) PFU of MV C- or MV V- showed significantly fewer and milder clinical symptoms and had a lower mortality. A total of 10(5) PFU V- virus were required to kill most YAC-CD46 mice, and less than half (44%) were killed with a corresponding dose of MV C-. Immunohistochemical staining for MV antigens showed similar extents of spread for MV C- and MV Ed but restricted spread for MV V- throughout the brain. Viral load and transcription were markedly reduced for V- but not for C-. Multiple cytokines and chemokines were equivalently upregulated for all three viruses. Therefore, MV C and V proteins encode virulence functions in vivo and likely operate via separate mechanisms.

Animals↗
PubMed2001

Chimeric subgroup A respiratory syncytial virus with the glycoproteins substituted by those of subgroup B and RSV without the M2-2 gene are attenuated in African green monkeys.

Using the existing reverse genetics system developed for the subgroup A respiratory syncytial virus (RSV), a chimeric virus (designated rA-G(B)F(B)) that expresses subgroup B-specific antigens was constructed by replacing the G and F genes of the A2 strain with those of the 9320 strain of subgroup B RSV. rA-G(B)F(B) grew well in tissue culture, but it was attenuated in the respiratory tracts of cotton rats and African green monkeys. To further attenuate this chimeric RSV, the M2-2 open reading frame was removed from rA-G(B)F(B). rA-G(B)F(B)DeltaM2-2 was highly attenuated in replication in the respiratory tracts of the infected monkeys, but it provided complete protection against wild-type subgroup B RSV challenge following two doses of infection. In this study, rA2DeltaM2-2 (a recombinant A2 RSV that lacks the M2-2 gene) was also evaluated in African green monkeys. The replication of rA2DeltaM2-2 was highly restricted in both the upper and lower respiratory tracts of the infected monkeys and it induced titers of serum anti-RSV neutralizing antibody that were slightly lower than those induced by wild-type rA2. When rA2DeltaM2-2-infected monkeys were challenged with wild-type A2 virus, the replication of the challenge virus was reduced by approximately 100-fold in the upper respiratory tract and 45,000-fold in the lower respiratory tracts. rA2DeltaM2-2 and rA-G(B)F(B)DeltaM2-2 could represent a bivalent RSV vaccine composition for protection against multiple strains from the two RSV subgroups.

Animals↗
PubMed2002

Human influenza a viral genes responsible for the restriction of its replication in duck intestine.

Although influenza A viruses are occasionally transmitted from one animal species to another, their host range tends to be restricted. Currently circulating human influenza A viruses are thought to have originated from avian viruses, yet none of these strains replicate in duck intestine, a major site of avian virus replication. Although the hemagglutinin (HA) and neuraminidase (NA) genes are known to restrict human virus replication in ducks, the contribution of the other viral genes remains unknown. To determine the genetic basis for host range restriction of the replication of human influenza A virus in duck intestine, we first established a reverse genetics system for generating A/Memphis/8/88 (H3N2) (Mem/88) and A/mallard/New York/6750/78 (H2N2) (Mal/NY) viruses from cloned cDNAs. Using this system, we then attempted to generate reassortant viruses with various combinations of candidate genes. We were able to generate single-gene reassortants, which possessed PB2, NP, M, or NS from Mem/88, with the remainder from Mal/NY. Despite unsuccessful production of other single-gene reassortants from Mem/88, we did generate reassortant viruses comprised of both the HA and the NA, all three polymerase genes (PB2, PB1, and PA), or all polymerase genes and the NP gene from Mem/88, with the rest derived from Mal/NY. Among these reassortants, only those possessing the M or NS gene from Mem/88 and the remainder from Mal/NY replicated in duck intestine. These results indicate incompatibility between the genes of avian and human influenza A viruses and indicate that all genes other than the M and NS restrict replication of human influenza A virus in duck intestine.

Animals↗
PubMed2005

Coronavirus reverse genetics by targeted RNA recombination.

Targeted RNA recombination was the first reverse genetics system devised for coronaviruses at a time when it was not clear whether the construction of full-length infectious cDNA clones would become possible. In its current state targeted RNA recombination offers a versatile and powerful method for the site-directed mutagenesis of the downstream third of the coronavirus genome, which encodes all the viral structural proteins. The development of this system is described, with an emphasis on recent improvements, and multiple applications of this technique to the study of coronavirus molecular biology and pathogenesis are reviewed. Additionally, the relative strengths and limitations of targeted RNA recombination and infectious cDNA systems are contrasted.

Coronavirus↗
PubMed2005

Recombinant rhabdoviruses: vectors for vaccine development and gene therapy.

The establishment of methods to recover rhabdoviruses from cDNA, so-called reverse genetics systems, has made it possible to genetically engineer rhabdoviruses and to study all aspects of the virus life cycle by introducing defined mutations into the viral genomes. It has also opened the way to make use of the viruses in biomedical applications such as vaccination, gene therapy, or oncolytic virotherapy. The typical gene expression mode of rhabdoviruses, a high genetic stability, and the propensity to tolerate changes in the virus envelope have made rhabdoviruses attractive, targetable gene expression vectors. This chapter provides an overview on the possibilities to manipulate biological properties of the rhabdoviruses that may be important for further development of vaccine vectors and examples of recombinant rhabdoviruses expressing foreign genes and antigens.

Animals↗
PubMed2027

Production of Viral Particles from a Chikungunya Virus Infectious Clone.

Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus, which poses challenges for its study and genetic manipulation. Because direct mutagenesis of viral RNA genomes is technically impractical, reverse genetics systems are essential tools for investigating viral biology. To enable such approaches, infectious clones containing a full-length cDNA copy of the viral genome are constructed. The cDNA is positioned under the control of a bacteriophage RNA polymerase promoter, allowing commercial RNA polymerases to use the linearized plasmid as a template for the in vitro transcription of full-length viral genomic RNA (gRNA). Importantly, positive-sense viral genomes serve as mRNAs for the translation of viral proteins in a cellular environment, meaning that these transcripts contain all the information required to initiate viral replication. Following transfection into permissive cultured cells, viral proteins are expressed, enabling genome replication and, ultimately, the recovery of infectious particles from the cell supernatant. Here, we describe a detailed procedure for generating CHIKV particles through plasmid linearization, in vitro transcription, and subsequent RNA transfection.

Chikungunya virus↗
PubMed2004

Reverse genetics of mononegavirales.

"Reverse genetics" or de novo synthesis of nonsegmented negative-sense RNA viruses (Mononegavirales) from cloned cDNA has become a reliable technique to study this group of medically important viruses. Since the first generation of a negative-sense RNA virus entirely from cDNA in 1994, reverse genetics systems have been established for members of most genera of the Rhabdo-, Paramyxo-, and Filoviridae families. These systems are based on intracellular transcription of viral full-length RNAs and simultaneous expression of viral proteins required to form the typical viral ribonucleoprotein complex (RNP). These systems are powerful tools to study all aspects of the virus life cycle as well as the roles of virus proteins in virus-host interplay and pathogenicity. In addition, recombinant viruses can be designed to have specific properties that make them attractive as biotechnological tools and live vaccines.

Animals↗
PubMed1995

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize.

A reverse genetic system for studying excision of the transposable element Ds1 in maize plants has been established previously. In this system, the Ds1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of the Ac element, excision of Ds1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences required in cis for excision of Ds1. The Ds1 element contains the Ac transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5' subterminal region). We showed that mutation of these motifs abolished completely the excision capacity of Ds1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3') subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.

Base Sequence↗
PubMed2002

NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells.

Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation.

Cells, Cultured↗
PubMed2002

A single amino acid substitution in 126-kDa protein of Pepper mild mottle virus associates with symptom attenuation in pepper; the complete nucleotide sequence of an attenuated strain, C-1421.

The complete nucleotide sequence of an attenuated Pepper mild mottle virus (PMMoV C-1421) RNA genome has been determined. There were two differences from the type isolate in Japan (PMMoV-J). The mutations were located in the middle of the 126-kDa protein (126 K) gene; one mutation influenced amino acid substitution at 649th Val to Ala (V649A), and the other was silent. The analyses using the reverse genetic system of PMMoV-J revealed that symptom attenuation on pepper related to V649A. Accumulations of 126 K and coat protein (CP) in V649A mutant-infected pepper were lower than those of PMMoV-J in immunoblotting. These results suggest that V649A substitution in 126 K affects the accumulation of 126 K leading to a limitation of CP accumulation.

Amino Acid Substitution↗
PubMed1992

Tn9 CAT gene contains a promoter for vaccinia virus transcription: implications for reverse-genetic techniques.

Vaccinia virus-dependent CAT expression was observed in virus-infected cells cotransfected with a promoterless CAT gene. Restriction endonuclease resection of the CAT plasmid indicated that expression was due to recognition by vaccinia virus RNA polymerase of sequences within the CAT gene itself, probably located within the 5' untranslated region of the gene. This observation is relevant to the design of reverse-genetic systems which use CAT as a reporter gene to detect replication of negative-strand RNA virus pseudogenomes.

Base Sequence↗
PubMed1993

Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions.

The neuraminidase (NA) of influenza viruses cleaves sialic acids from receptors, prevents self-aggregation and facilitates release of virus during budding from host cells. Although the structure and function of the globular head of the influenza virus NA has been well studied, much less is known about the stalk of the NA, the region between the viral membrane and the globular head. Applying a reverse genetics system, we altered the stalk of the influenza A/WSN/33 virus NA by making deletions, insertions and mutations in this region of the gene. Our data show that the length of the NA stalk can be variable. Deletions of up to 28 amino acids and insertions of up to 41 amino acids in the stalk region did not abolish formation of infectious progeny virus. The data also indicate that the cysteine at position 76 is essential for formation of infectious virus, and that deletions beyond the cysteine did not result in infectious virus. Interestingly, shortening of the length of the stalk region by 28 amino acids resulted in a virus with a markedly reduced growth rate in MDCK cells as compared to that in MDBK cells. An insertion of 41 extra amino acids into the stalk did not significantly interfere with viral growth in MDCK or MDBK cells, which suggests that the stalk region would tolerate the introduction of long foreign sequences.

Amino Acid Sequence↗
PubMed2006

Ebola virus: unravelling pathogenesis to combat a deadly disease.

Ebola virus (EBOV) causes severe haemorrhagic fever leading to up to 90% lethality. Increasingly frequent outbreaks and the placement of EBOV in the category A list of potential biothreat agents have boosted interest in this virus. Furthermore, development of new technologies (e.g. reverse genetics systems) and extensive studies on Ebola haemorrhagic fever (EHF) in animal models have substantially expanded the knowledge on the pathogenic mechanisms that underlie this disease. Two major factors in EBOV pathogenesis are the impairment of the immune response and vascular dysfunction. Here, we attempt to summarize the current knowledge on EBOV pathogenesis focusing on these two factors and on recent progress in the development of vaccines and potential therapeutics.

Animals↗
PubMed2004

Recombinant replication-restricted VSV as an expression vector for murine cytokines.

Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus that rapidly and efficiently shuts down the production of host cell-encoded proteins and utilizes the cell's protein production machinery to express high levels of virally encoded proteins. In an effort to take advantage of this characteristic of VSV, we have employed a reverse genetics system to create recombinant forms of VSV encoding a variety of murine cytokines. Previous studies have revealed that cells infected with recombinant VSV that lack expression of the surface glycoprotein (G protein), designated deltaG-VSV, more efficiently express and secrete recombinant proteins than do recombinant "wild-type" VSV. Therefore, murine cytokine-expressing recombinants were produced as deltaG viruses. Propagation of these deltaG viruses in cells that transiently express G protein in vitro results in G-complemented virions that can infect cells, shut down host protein synthesis, and express at high levels each virally encoded protein (including the designated cytokine). We assessed the ability of each deltaG-VSV construct to express recombinant cytokine by infecting BHK cells and then monitoring/measuring the production of the desired cytokine. When possible, the bioactivity of the cytokine products was also measured. The results presented here reveal that large quantities of bioactive cytokines can be produced rapidly and inexpensively using deltaG-VSV as a protein expression system.

Animals↗
PubMed2004

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes.

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.

Animals↗