The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys 3 -His 1 motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using ␤-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys 3 -His 1 motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys 3 -His 1 motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Human respiratory syncytial virus (hRSV) is an enveloped nonsegmented negative-strand RNA virus classified in the genus Pneumovirus of the family Paramyxoviridae (19). The genomic RNA of hRSV A2 strain is 15,222 nucleotides (nt) in length and encodes 11 proteins from 10 genes in the following gene order: 3Ј NS1-NS2-N-P-M-SH-G-F-M2-L 5Ј. Each gene transcription unit is flanked by highly conserved gene-start and gene-stop sequences and is monocistronic except for the M2 gene, which encodes two proteins unique to pneumoviruses, M2-1 and M2-2 (6, 7, 16). As with other negative-strand RNA viruses, synthesis of viral RNA requires a genomic RNA encapsidated with the nucleoprotein (N) along with the virusencoded phosphoprotein (P) and the large (L) polymerase protein (12,29). In addition, the M2-1 protein is also required for synthesis of RSV RNA. The M2-1 protein is an antiterminator that prevents premature termination during transcription (6, 10, 11) and enhances read-through transcription at gene junctions (13-15).The M2 mRNAs of all pneumoviruses encode two open reading frames (ORFs) that overlap at a similar location but with different overlapping sequences (1, 8). The M2-1 of hRSV A2 strain utilizes approximately 70% of the entire coding capacity of the M2 mRNA. The second ORF is located towards the 3Ј end of the mRNA and overlaps M2-1 by 4, 8, or 10 amino acids, depending on the initiation codon(s) used for translating M2-2. It has been proposed that the translation of hRSV M2-2 occurs by a mechanism that involves reverse translocation of ribosomes terminating at the first downstream M2-1 stop codon (1). The M2-2 protein is dispensable for RSV replication, and present data indicate that M2-2 is involved in regulating the switch between viral RNA transcription and replication (3, 17).The M2-1 protein of hRSV A2 strain is 194 amino acids in length, with a molecular weight of approximately 22,150 (6, 7). It contains a Cys 3 -His 1 motif in the N terminus from residues 7 to 25 that is highly conserved among human, bovine, ovine, and murine strains of pneumoviruses (1, 2, 29). Nuclear magnetic resonance spectroscopy and zinc back-titration analyses of an analo...

Restricted replication in the respiratory tract of rhesus monkeys is an intrinsic property of bovine parainfluenza virus type 3 (bPIV-3) strains. This host range phenotype of bPIV-3 has been utilized as a marker to evaluate the attenuation of bPIV-3 vaccines for human use. Two safety, immunogenicity and efficacy studies in primates evaluated and compared three human parainfluenza virus type 3 (hPIV-3) vaccine candidates: biologically derived bPIV-3, a plasmid-derived bPIV-3 (r-bPIV-3) and a chimeric bovine/human PIV-3 (b/hPIV-3). These studies also examined the feasibility of substituting Vero cells, cultured in the presence or absence of foetal bovine serum, for foetal rhesus lung-2 (FRhL-2) cells as the tissue culture substrate for the production of bPIV-3 vaccine. The results demonstrated that (i) Vero cell-produced bPIV-3 was as attenuated, immunogenic and efficacious as bPIV-3 vaccine grown in FRhL-2 cells, (ii) plasmid-derived bPIV-3 was as attenuated, immunogenic and efficacious as the biologically derived bPIV-3 and (iii) the b/hPIV-3 chimera displayed an intermediate attenuation phenotype and protected animals completely from hPIV-3 challenge. These results support the use of bPIV-3 vaccines propagated in Vero cells in human clinical trials and the use of b/hPIV-3 as a virus vaccine vector to express foreign viral antigens. The bPIV-3 vaccine strain (bPIV-3/Kansas/15626/84) used here was isolated in 1984 from a calf with pneumonia in Kansas and was shown to be immunogenic in hamsters (Haller et al., 2000) and in Old World and New World non-human primates (van Wyke Coelingh et al., 1988). Compared to hPIV-3, bPIV-3 was approximately 100-to 1000-fold restricted for replication in the lower (LRT) and S. Pennathur and A. A. Haller contributed equally to this work.

Clustered charge-to-alanine mutagenesis was performed on the large (L) polymerase protein of human respiratory syncytial virus to identify charged residues in the L protein that are important for viral RNA synthesis and to generate temperature-sensitive viruses. Clusters of three, four, and five charged residues throughout the entire L protein were substituted with alanines. A minigenome replicon assay was used to determine the functions of the mutant L proteins and to identify mutations that caused temperature sensitivity by comparing the level of reporter gene expression at 39 and 33 degrees C. Charge-to-alanine mutations were introduced into an antigenomic cDNA derived from RSV A2 strain to recover infectious viruses. Of the 27 charge-to-alanine mutations, 17 recombinant viruses (63%) were obtained. Seven mutants (41%) exhibited small plaque morphologies and/or temperature-sensitive growth in tissue culture. To generate mutant viruses with more temperature-sensitive and attenuated phenotypes, several clusters of charge-to-alanine substitutions were combined. Five combination mutants were recovered that exhibited shut-off temperatures ranging from 36 to 39 degrees C in tissue culture and restricted replication in the respiratory tracts of cotton rats.

RNAse mimics are small molecules that can cleave RNA in a fashion similar to ribonucleases. These compounds would be very useful as gene specific reagents if their activities could be regulated and targeted. We demonstrate here that polyamides with methionine substituents show enhanced RNA cleavage activity relative to other polyamides. Conjugation of these compounds to aminoglycosides produced RNAse mimics that are capable of inhibiting eukaryotic protein synthesis. As a new class of compounds capable of interacting with nucleic acids, these novel aminoglycoside-polyamides constitute promising scaffolds for the construction of nuclease mimics with biological activity.