GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Polymerases of paramyxoviruses and pneumoviruses.

The paramyxo- and pneumoviruses are members of the order Mononegavirales, a group of viruses with non-segmented, negative strand RNA genomes. The polymerases of these viruses are multi-functional complexes, capable of transcribing subgenomic capped and polyadenylated mRNAs and replicating the genome. Although there is no native structure available for any complete paramyxo- or pneumovirus polymerase, functional and structural studies of a fragment of a pneumovirus polymerase protein and mutation analyses and resistance profiling of small-molecule inhibitors have generated a wealth of mechanistic information. This review integrates these data with the structure of a related polymerase, identifying similarities, differences, gaps in knowledge, and avenues for antiviral drug development.

Independent structural domains in paramyxovirus polymerase protein.

All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases.

Effects of multiple amino acids of the parainfluenza virus 5 fusion protein on its haemagglutinin-neuraminidase-independent fusion activity.

The fusion (F) protein of parainfluenza virus 5 (PIV-5) strain W3A is able to induce cell fusion when it is expressed alone in baby hamster kidney cells, whilst the F protein of PIV-5 strain WR induces cell fusion only when co-expressed with the haemagglutinin-neuraminidase (HN) protein. It has been shown previously that when Leu-22 of the WR F protein is replaced with the W3A F counterpart (Pro-22), the resulting mutant L22P exhibits HN-independent fusion activity. Furthermore, previous chimeric analysis between L22P and the F protein of PIV-5 strain T1 has suggested that Glu-132 also contributes to the HN-independent fusion activity of L22P. It was shown here that substitution of Glu-132 of L22P with various amino acids including the T1 F protein counterpart (Lys-132) resulted in a reduction in fusion activity, whereas substitution with Asp was the exception in being tolerated. Interestingly, reduced fusion activity of an L22P mutant that harboured the E132K substitution could be restored by an additional D416K substitution but not by a D416E mutation, suggesting that the presence of the same charge at positions 132 and 416 is important for the HN-independent fusion activity. In contrast, substitution of Leu-22 of the WR F protein with various amino acids except those with aliphatic side chains resulted in acquisition of fusion activity, suggesting that the HN dependence of the WR F protein in the induction of cell fusion is attributable to the hydrophobicity of Leu-22. These results indicate that at least three amino acids are involved in the HN-independent fusion activity of the PIV-5 F protein.

High throughput screening assay for negative single stranded RNA virus polymerase inhibitors.

The Paramyxoviridae form a large family of viruses containing many human and veterinary pathogens for which a need for antiviral treatment is emphasized, particularly following the recent emergence of new viruses. The viral RNA-dependent RNA polymerase constitutes an obvious target for antiviral compounds. An in vitro assay was developed that allows high throughput screening of compounds potentially inhibiting the Sendai virus RNA-dependent RNA polymerase. Screening relies on the detection of the Photinus pyralis luciferase produced in a transcription/translation coupled assay using a mini-replicon virus. It contains an internal control for possible adverse effects of the tested compounds on translation or on luciferase activity. It is estimated that the mini-replicon template produced in one fertilized egg is sufficient to run 5000-10,000 reactions. This assay constitutes a simple, sensitive and easily automated method to perform high throughput screening of Paramyxoviridae RNA-dependent RNA polymerase inhibitors.

Role of cellular kinases in the gene expression of nonsegmented negative strand RNA viruses.

Nonsegmented negative strand RNA viruses package an RNA-dependent RNA polymerase composed of two subunits, a large protein L and a phosphoprotein P, for transcription and replication of their genome RNAs. The RNA polymerase activity resides within the L protein, while the P protein acts as a transcription factor or transactivator of the polymerase. Since P protein is heavily phosphorylated and phosphorylation is known to regulate function of many viral as well as cellular proteins, the role of phosphorylation of P protein in the gene expression of this group of RNA viruses has recently been investigated. Through expression in bacteria the P protein was produced in large quantity in the nonphosphorylated form and involvement of cellular kinase(s) in its phosphorylation was studied. Casein kinase II and/or protein kinase C have been shown to play a critical role in the activation of P protein in transcription. These findings have opened up a new avenue for studying an important regulatory step in virus gene expression that may lead to the development of an effective antiviral agent.

Comparison of the polymerases (L genes) of spring viremia of carp virus and infectious hematopoietic necrosis virus.

We have determined the partial nucleotide sequences of the polymerase genes of the fish rhabdoviruses, spring viremia of carp virus (SVCV) and infectious hematopoietic necrosis virus (IHNV). At this point we have deduced the amino acid sequences and analysed the first 1,400 amino acids comprising two thirds of the polymerase genes of SVCV and IHNV. We have compared sequence similarities of SVCV and IHNV polymerases with other rhabdovirus and paramyxovirus polymerases. The SVCV polymerase showed the closest relationship with the vesicular stomatitis virus polymerases and also shared significant sequence identity with the polymerase of rabies virus. Other rhabdovirus and paramyxovirus polymerases showed lower sequences identities with the SVCV polymerase. The IHNV polymerase shared a relatively low amino acid sequence identity with the rabies virus polymerase, and similar low identities with other rhabdovirus and paramyxovirus polymerases. Several domains of various lengths were conserved in the virus polymerases included in this study. These domains were less conserved in the IHNV polymerase than in the SVCV polymerase, and some of the domains present in the other polymerases were not identified in the IHNV. These preliminary results indicate that SVCV is closely related to mammalian vesiculoviruses and that IHNV may be only distantly related to mammalian lyssa and vesiculotype rhabdoviruses.

Sequence similarity between Borna disease virus p40 and a duplicated domain within the paramyxovirus and rhabdovirus polymerase proteins.

We report the sequence of a Borna disease virus clone (pBDV-40) that encodes a 40-kDa protein (p40) found in the nuclei of infected cells. Comparative sequence analysis indicates that p40 is distantly similar to two different regions in the L-polymerase proteins encoded by paramyxoviruses and rhabdoviruses. The p40 sequence similarity indicates a previously undetected duplication in these viral polymerases. Phylogenetic reconstruction suggests that the gene that encodes p40 last shared a common ancestor with these viral polymerase genes prior to the duplication event. These findings support the hypothesis that Borna disease virus is a negative-strand RNA virus and suggest that p40 is involved in transcription and/or replication. The discovery of a duplication within the polymerase proteins of paramyxoviruses and rhabdoviruses has profound implications for the mapping of enzymatic activities within these multifunctional proteins.

Folding and oligomerization properties of a soluble and secreted form of the paramyxovirus hemagglutinin-neuraminidase glycoprotein.

The paramyxovirus SV5 hemagglutinin-neuraminidase (HN) glycoprotein (a type II integral membrane protein) was converted into a soluble and secreted form (HN-F) by replacing the HN signal/anchor domain with a hydrophobic domain that can act as a cleavable signal sequence. Approximately 40% of the HN-F synthesized was secreted from cells (t1/2 approximately 2.5-3 hr). The extracellular HN-F molecules were identified as disulfide-linked dimers and the majority of the population of molecules were resistant to endoglycosidase H digestion. Examination of the oligomeric form of the secreted HN-F, by sucrose density gradient sedimentation, indicated that under conditions where HN was a tetramer, HN-F was found to be a dimer, and no extracellular HN-F monomeric species could be detected. Secreted HN-F was fully reactive with conformation-specific monoclonal antibodies and was enzymatically active as shown by HN-F having neuraminidase activity. Examination of the intracellular HN-F species indicated that HN-F monomers were slowly converted to the disulfide-linked form and that under the sucrose density gradient sedimentation conditions used the HN-F monomers aggregated. Some of the HN-F monomers were degraded intracellularly. These data are discussed in relationship to the seemingly different folding and oligomerization requirements for the intracellular transport of soluble and membrane bound forms of a glycoprotein. The soluble and biologically active form of HN may be suitable for further structural and enzymatic studies.

Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains.

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

Further evidence of the circulation of PMV-4 and influenza viruses with N2-1957 enzyme in the migratory waterfowls.

In the years 1980-1984, one paramyxovirus type 4 and 11 influenza viruses were isolated from cloacal swabs collected from migratory waterfowls in Fed. Rep. Germany. One influenza virus of H4N8 subtype was isolated from swabs of commercial ducks collected at an abbatoir. Seven of 10 influenza strains, isolated from mallard ducks and coot were identified as a mixture of 2-3 strains of H1, H4, and H5 subtype; 3 virus strains from the same locality relate antigenically to subtype H4 with enzyme serologically identical with N2--Singapore/57 as demonstrated by means of polyclonal and monoclonal antibody.

Monoclonal antibodies against avian paramyxovirus-3: antigenic mapping and functional analysis of the haemagglutinin-neuraminidase protein and the characterization of nonspecific monoclonal antibodies.

Sixteen monoclonal antibodies (Mabs) which immunoprecipitated the haemagglutinin neuraminidase (HN) of chorio-allantoic membrane-grown avian paramyxovirus-3 (PMV-3) of British turkeys were produced. Thirteen were PMV-3 specific. Three were nonspecific because they also bound to other viral proteins and to bovine kidney cells treated with neuraminidase enzyme or infected with influenza virus. The thirteen specific Mab defined four antigenic regions A-D by competition and variant selection assays. Region A was subdivided into five epitopes and region B into two epitopes. The thirteen Mab neutralized and were active in haemagglutination inhibition (HI) and twelve were active in haemolysis inhibition (HLI) tests. Neuraminidase inhibition (NI) was epitope-dependent. Mabs to five of the epitopes A1, A2, A3, A5, and C bound to the 1981 British turkey isolates but not the 1968 American turkey isolate. The Mab to epitope A4 bound to both viruses. The Mabs to epitopes B1, B2, and D also bound to a parakeet isolate of PMV-3 which was the third PMV-3 tested. The Mab to B2 gave identical titres to all three viruses and had HI, HLI, and NI activities. This made it a potential diagnostic reagent for avian PMV-3 viruses. One of the nonspecific Mabs bound to lactose-like moieties as reported on influenza virus and one to maltose-like moieties as on retroviruses. Immunoglobulin from all three nonspecific Mab had some HI activity.

Antigenic relationships between avian paramyxoviruses. I. Quantitative characteristics based on hemagglutination and neuraminidase inhibition tests.

Comprehensive hemagglutination inhibition (HI) and neuraminidase inhibition (NI) cross reaction tests were performed using 8 of 9 serotypes of avian paramyxoviruses (PMV). The studies were designed as full scale repeating experiments which permitted an adequate statistical treatment and elaboration of quantitative criteria of antigenic kinship. The results have shown diverse antigenic relationships between different avian paramyxovirus (PMV) serotypes which were asymmetric in some cases. The antigenic relationships found by HI test did not always parallel those found by NI tests. The antigenic inter-relationships have been displayed quantitatively in a diagram. This has given a basis for some suggestions concerning: the independent antigenic drift of the HA and Nase antigenic sites of hemagglutinin-neuraminidase (HN) glycoprotein of avian PMVs; a tentative subdivision of the whole group of avian PMVs into two subgroups: the first including PMV-2 and PMV-6 serotypes and the second including PMV-1, PMV-3, PMV-4, PMV-7, PMV-8 and PMV-9 serotypes; the conception that genomic material coding for the HN glycoprotein consists of a "common-to-all-the-PMVs" portion and a "serotype-specific" portion, on one hand, and of a "conserved" portion and a "variable" portion, on the other; the ratios between the portions have been shown to be different for, at least, certain PMV serotypes; the evolutionary pathways of the avain PMV HN antigenic drift.

Biochemical properties of paramyxovirus Duck/Mississippi/75 neuraminidase.

The neuraminidase activity of two strains of Duck/Mississippi/75 virus:DK/Mississippi/320 and DK/Mississippi/334 was studied. These neuraminidases hydrolyse the alpha 2 leads to 3 and alpha 2 leads to 8 ketosidic bonds of different substrates such as fetuin, N-acetyl neuramine lactose and colominic acid, but do not hydrolyse the alpha 2 leads to 6 bonds of mucin type I and type II. The kinetic values of the neuraminidases, Michaelis constant, maximal and initial velocities and the effect of pH, temperature and detergents were also evaluated. The isolates differ mainly in the optimal pH and temperature conditions of activity. As with other paramyxovirus neuraminidases, the enzyme of DK/Mississippi/75 was destroyed by ionic but not non-ionic detergents.