We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 103 plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 104–5 × 107 pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

https://www.pnas.org/content/pnas/96/16/9345.full.pdf

Generation of influenza A viruses entirely from cloned cDNAs

▪ Abstract ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.

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RNA Editing by Adenosine Deaminases That Act on RNA

In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all cis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.

Measles virus continues to be a major killer of children, claiming roughly one million lives a year Measles virus infection causes profound immunosuppression, which makes measles patients susceptible to secondary infections accounting for high morbidity and mortality The Edmonston strain of measles virus, and vaccine strains derived from it, use as a cellular receptor human CD46 (refs 3, 4), which is expressed on all nucleated cells; however, most clinical isolates of measles virus cannot use CD46 as a receptor Here we show that human SLAM (signalling lymphocyte-activation molecule; also known as CDw150), a recently discovered membrane glycoprotein expressed on some T and B cells, is a cellular receptor for measles virus, including the Edmonston strain Transfection with a human SLAM complementary DNA enables non-susceptible cell lines to bind measles virus, support measles virus replication and develop cytopathic effects The distribution of SLAM on various cell lines is consistent with their susceptibility to clinical isolates of measles virus The identification of SLAM as a receptor for measles virus opens the way to a better understanding of the pathogenesis of measles virus infection, especially the immunosuppression induced by measles virus

SLAM (CDw150) is a cellular receptor for measles virus.

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.

Rescue of Influenza A Virus from Recombinant DNA

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.

Rescue of measles viruses from cloned DNA.

Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm.

Domains of the Measles Virus N Protein Required for Binding to P Protein and Self-Assembly

Subacute sclerosing panencephalitis (SSPE), a very rare but lethal disease caused by measles viruses (MV) persisting in the human central nervous system (CNS) is characterized by lack of viral budding, reduced expression of the viral envelope proteins and spread of MV genomes through the CNS despite massive immune responses. The five major MV genes from several SSPE cases were cloned and sequenced, the two transmembrane envelope glycoproteins hemagglutinin (H) and fusion protein (F) were expressed and their maturation, cellular localization and functionality analyzed. We conclude that 1) mutations in the MV genes arise not only individually, by errors of the MV polymerase, but also in clusters as hypermutations, presumably due to RNA unwinding/modifying activity altering accidentally formed double-stranded RNA regions, 2) MVs spread in SSPE brains after clonal selection, 3) the MV matrix (M) gene is most heavily mutated and dispensable, 4) the two genes encoding envelope transmembrane proteins give rise to functional but altered proteins (typically F is heavily altered in its cytoplasmic domain), 5) H protein is transported poorly to the cell surface, 6) F and H proteins maintain tightly interdepending fusion functions, presumably to allow local cell fusion and MV ribonucleoprotein (RNP) spread through the CNS.

Generation and properties of measles virus mutations typically associated with subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.

M. Huber

Papers

Rescue of measles viruses from cloned DNA.

Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm.

Domains of the Measles Virus N Protein Required for Binding to P Protein and Self-Assembly

Generation and properties of measles virus mutations typically associated with subacute sclerosing panencephalitis.

Functional and nonfunctional measles virus matrix genes from lethal human brain infections.