Kyosuke Nagata

Viral RNA polymerases

Recent progress in molecular biological techniques revealed that genomes of animal viruses are complex in structure, for example, with respect to the chemical nature (DNA or RNA), strandedness (double or single), genetic sense (positive or negative), circularity (circle or linear), and so on. In agreement with this complexity in the genome structure, the modes of transcription and replication are various among virus families. The purpose of this article is to review and bring up to date the literature on viral RNA polymerases involved in transcription of animal DNA viruses and in both transcription and replication of RNA viruses. This review shows that the viral RNA polymerases are complex in both structure and function, being composed of multiple subunits and carrying multiple functions. The functions exposed seem to be controlled through structural interconversion.

Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system

Influenza virus RNA polymerase catalyzes multiple step reactions in transcription and replication of the genome RNA. The core enzyme is composed of each one of the three P proteins, PB1, PB2 and PA (Honda et al. (1990) J. Biochem. 107, 624-628). For detailed analysis of the role of each P protein and of the functional domains on each P polypeptide, we expressed individual P proteins in cultured insect cells after infection with recombinant baculoviruses. PB1 and PB2 accumulated in cell nuclei whereas PA stayed in cytoplasm. Both the PB1 and PB2 proteins were purified from aggregates in the respective nuclear extract, and the PA was partially purified from the cytoplasm. RNA polymerase was reconstituted by mixing the three P proteins in a urea solution and then dialyzing against a reconstitution buffer. The reconstituted enzyme was able to transcribe model RNA templates. Minus-sense RNA was a better template than plus-sense RNA.

Enhancer of human polyoma JC virus contains nuclear factor I-binding sequences; analysis using mouse brain nuclear extracts

Neurotrophic human JC virus carries a 98 bp duplicate enhancer responsible for tissue-specific gene expression. DNase I footprinting studies using mouse brain nuclear extracts revealed weak (pseudo NFI motif) and strong (NFI motif) nuclear factor I-binding sequences just upstream (at 229) and in the middle (at 156 and 58) of the enhancer, respectively. In vitro transcription driven in brain extracts demonstrated that the NFI motif is a possible transcription control element. Together with previous observations (Khalili, K., Rappaport, J. and Khoury, G. (1988) EMBO J. 7, 1205-1210 (20], the NFI motif is suggested to play an important role in early gene expression of JCV.

Inhibition of influenza virus infection by pine cone antitumor substances

The anti-influenza virus activity of polysaccharides and other high molecular weight fractions from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. was investigated. None of the fractions affected the growth of MDCK cells. The acidic PCE substances markedly suppressed the growth of the influenza virus in MDCK cells. Significant inhibition of both the viral protein synthesis in infected cells and virion-associated RNA-dependent RNA polymerase activity was observed with these acidic fractions. Although amantadine inhibited virus plaque formation as effectively as PCE fractions, it was less effective in inhibiting the RNA polymerase activity. These results suggest that PCE, which has been shown to contain antitumor substance(s), also contains anti-influenza virus substance(s).

Possible involvement of lignin structure in anti-influenza virus activity

Commercial lignins suppressed the growth of influenza A virus infecting MDCK cells, and the RNA-dependent RNA synthesis, as efficiently as the high-molecular weight fractions extracted from pine cone of Pinus parviflora Sieb. et Zucc. The anti-influenza A virus activity of both pine cone extract and commercial alkali-lignin was considerably reduced by treatment with sodium chlorite, but was not affected by sulfuric acid or trifluoroacetic acid. The degraded components of lignin, various synthesized polyphenols unrelated to lignin, and natural and chemically modified glucans, were not appreciably inhibitory. The data suggest that the polymerized phenolic structure of lignified materials is responsible for the anti-influenza A virus activity.

Monoclonal antibody analysis of influenza virus matrix protein epitopes involved in transcription inhibition

Influenza virus M1 protein has been shown to inhibit viral RNA transcription, and in this study the epitopes on M1 critical for this function were localized. When a battery of 15 monoclonal anti-M1 antibodies were reacted with chemically cleaved fragments of M1 on a western blot, five distinct banding patterns were observed. A representative antibody was selected from each banding group, and its ability to reverse M1-effected transcription inhibition was measured. From these data, the sites on M1 critical for transcription inhibition were deduced. It appears now that the regions on M1 in the vicinity of amino acid residues ♯70 and ♯140 are critical for inhibition. Furthermore, by taking into account the hydropathicity and secondary structure, it is hypothesized that amino acids ♯70 and ♯140 are physically close together in the final three-dimensional conformation of M1 protein and that the residues in between form a loop and are thus removed from the functional site.

Mechanism of influenza virus transcription inhibition by matrix (M1) protein

The mechanism by which influenza virus matrix (M1) protein inhibits viral RNA (vRNA) transcription was investigated. Evidence has been generated that M1 protein inhibits the steps of vRNA transcription initiation and reinitiation more effectively than that of RNA chain elongation. The vRNA-associated nucleocapsid protein (NP) appears to be critical for this inhibition, implying that M1 protein binds to the ribonucleoprotein complex (RNP) through NP.

Nuclear factor I represses the reverse-oriented transcription from the adenovirus type 5 DNA terminus

The promoter activity of a cloned inverted terminal repeat (ITR) of human adenovirus (Ad) type 5 was found to be oriented in the opposite direction with respect to that of DNA replication in a cell-free transcription system using HeLa nuclear extracts. The major transcript was initiated outside the Ad sequence about 30 nucleotides downstream from the putative TATA-box located between nucleotide positions 9-18 of Ad 5 left terminus. Competitive transcription experiments using double-stranded oligonucleotides and isolated nuclear factor I revealed that nuclear factor I and its cognate binding site located upstream of the putative TATA-box are involved in negative regulation of the transcription from ITR promoter.

Anti-influenza virus activity of synthetically polymerized phenylpropenoids

Various dehydrogenation polymers (the so-called synthetic lignins) were synthesized from four different phenylpropenoids by endwise and bulk polymerizations and investigated for their anti-influenza virus activity. All of these materials suppressed the plaque formation of influenza virus infecting MDCK cells and the RNA-dependent RNA synthesis as effectively as the high molecular weight lignin-related extract (Fr. VI) of pine cone of Pinus parviflora Sieb. et Zucc. The structural simplicity and higher solubility of the synthesized polymers suggest that they might be regarded as potential candidates for medicinal antiviral resources.

Enzymatic properties of the mouse Mx1 protein-associated GTPase

Murine Mx1 protein, an interferon-inducible nuclear protein present in inbred mouse Mx+ strains, confers resistance to influenza virus infection. The purified Mx1 protein was found to carry the activities of both GTPase and GTP-binding. Enzymatic properties of the Mx1-associated GTPase were examined using the Mx1 protein purified from Escherichia coli expressing Mx1 cDNA. The Mx1 protein exhibited a substrate preference for GTP. The Vmax of ATP hydrolysis was about 7.6% the rate of GTP hydrolysis. The hydrolysis of CTP and UTP was virtually negligible. The Km for GTP hydrolysis was 667 microM and the rate was 13.8 mol GTP hydrolysis per min per mol Mx1 protein. The enzymatic properties of Mx1 protein-associated GTPase were compared with those of the GTPase super-gene family and the Mx-related family.