Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up The ability to recover infectious virus from cDNA clones of RNA virus genomes facilitates molecular analyses of every aspect of the viral replicative cycle. For example, the demonstration that a plasmid encoding bacteriophage Q13 RNA gave rise to infectious phage when introduced into Escherichia coli allowed the experimental manipulation of the genomes of Qf3 and other RNA phages (1). The subsequent finding that a cDNA clone of the entire genome of poliovirus gave rise to infectious virus particles when introduced into mammalian cells in culture extended these developments to animal viruses (2). Since then, infectious cDNA clones of a variety of positivesense RNA virus genomes have been constructed, permitting the application of recombinant DNA technology to all aspects of the biology of these viruses (for a review, see ref.3). In contrast to this success with positive-strand RNA viruses, attempts to generate infectious cDNA clones of negativestrand RNA viruses have until recently been unsuccessful. The genomes of negative-strand RNA viruses must be associated with their nucleocapsid protein to form the active ribonucleoprotein template for their RNA-dependent RNA polymerase (4). As a consequence of this and in contrast to positive-strand RNA viruses, naked genomic RNAs of negative-strand viruses are not infectious. Therefore the development of a system in which cDNA transcripts could be associated with the nucleocapsid protein and the viral polymerase was a prerequisite for the successful manipulation of the genomes of negative-strand RNA viruses. Several developments have permitted the manipulation of incomplete genomes of some of these viruses (5-15) and have led to the generation of infectious rabies virus from a positive-sense transcript of a complete genome-length cDNA clone (16).Working with the prototypic nonsegmented negative-strand RNA virus, vesicular stomatitis virus (VSV), our laboratory developed a system for the recovery of infectious defective interfering (DI) particles and VSV subgenomic replicons from cDNA clones (12,17). In these experiments the production of infectious particles was supported entirely by viral proteins expressed in cells from plasmid vectors. These vectors were transcribed following transfection into cultured baby hamster kidney (BHK21) cells that were infected with the vaccinia virus (VV)-T7 RNA polymerase recombinant ref. 18) to provide a source of transcriptase. Negative-sense RNAs were generated from transcription plasmids that encoded subgenomic analogs or naturally occurr...

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The family Bunyaviridae of segmented, negative-stranded RNA viruses includes over 350 members that infect a bewildering variety of animals and plants. Many of these bunyaviruses are the causative agents of serious disease in their respective hosts, and are classified as emerging viruses because of their increased incidence in new populations and geographical locations throughout the world. Emerging bunyaviruses, such as Crimean-Congo hemorrhagic fever virus, tomato spotted wilt virus and Rift Valley fever virus, are currently attracting great interest due to migration of their arthropod vectors, a situation possibly linked to climate change. These and other examples of continued emergence suggest that bunyaviruses will probably continue to pose a sustained global threat to agricultural productivity, animal welfare and human health. The threat of emergence is particularly acute in light of the lack of effective preventative or therapeutic treatments for any of these viruses, making their study an important priority. This review presents recent advances in the understanding of the bunyavirus life cycle, including aspects of their molecular, cellular and structural biology. Whilst special emphasis is placed upon the emerging bunyaviruses, we also describe the extensive body of work involving model bunyaviruses, which have been the subject of major contributions to our overall understanding of this important group of viruses.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.H uman respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children and the immunocompromised. HRSV is a pneumovirus of the Paramyxoviridae family of the order Mononegavirales-the nonsegmented negative-strand RNA viruses. Its genome encodes 10 genes that are each transcribed by an RNA-dependant RNA polymerase (RdRp) into single mRNAs. During transcription, the RdRp uses a single promoter in the 3′ leader region (Le) of the genome (1) and responds to gene start and gene end sequences flanking each gene, directing initiation and termination of mRNA transcription, respectively (2). During genome replication, the RdRp bypasses these signals to synthesize a full-length antigenome. The virus-encoded components needed for RNA replication are the large protein (L), the nucleocapsid protein (N), and the phosphoprotein (P). However, complete transcription of mRNAs also requires the M2-1 transcription antiterminator protein (3, 4).M2-1 prevents premature transcription termination both intra-and intergenically (5, 6). M2-1 is essential for HRSV multiplication although it is not currently known how M2-1 effects its role, and deciphering this role is complicated by its multiple interactions with other viral components, namely P (7, 8), RNA (9), and the matrix protein (M) (10). M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11). Based on mutational analysis and a partial M2-1 structure determined using NMR (12, 13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exh...