Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides

Abstract

We have undertaken by biochemical and immunological experiments to locate the region of the matrix (M,) protein responsible for down-regulating endogenous transcription of A/WSNI33 influenza virus. A more refined map of the antigenic determinants of the M, protein was obtained by binding of epitope-specific monoclonal antibodies (MAbs) to chemically cleaved fragments. Epitope 2-specific MAb 289/4 and MAb 7E5 reverse transcription inhibition by M, protein and react with a 4-kilodalton cyanogen bromide fragment extending from amino acid Gly-129 to Gln-164. Anti-idiotype serum immunoglobulin G prepared in rabbits immunized with MAb 289/4 or MAb 7E5 mimicked the action of M, protein by inhibiting transcription in vitro of influenza virus ribonucleoprotein cores. This transcription-inhibition activity of anti-MAb 7E5 immunoglobulin G and anti-MAb 289/4 immunoglobulin G could be reversed by MAb 7E5 and MAb 289/4 or could be removed by MAb 7E5-Sepharose affinity chromatography. Transcription of influenza virus ribonucleoprotein was inhibited by one of three synthetic oligopeptides, a nonodecapeptide SP3 with an amino acid sequence corresponding to Pro-90 through Thr-108 of the Ml protein. Of all the structural proteins of influenza virus, only NP and Ml showed strong affinity for binding viral RNA or other extraneous RNAs. The 4-kilodalton cyanogen bromide peptide (Gly-129 to Gln-164), exhibited marked affinity for viral RNA, the binding of which was blocked by epitope 2-specific MAb 7E5 but not by MAbs directed to three other epitopes. Viral RNA also bound strongly to the nonodecapeptide SP3 and rather less well to anti-idiotype anti-MAb 7E5; these latter viral RNA-binding reactions were only slightly blocked by preincubation of anti-MAb 7E5 or SP3 with MAb 7E5. These experiments suggest the presence of at least two RNA-binding sites, which also serve as transcription-inhibition sites, centered around amino acid sequences 80 through 109 (epitope 4?) and 129 through 164 (epitope 2) of the 252 amino acid M1 protein of A/WSN/33 influenza virus. A hydropathy plot of the Ml protein calculated by free-energy transfer suggests that the two hydrophilic transcription-inhibition RNA-binding domains are brought into close proximity by an a-helix-forming intervening hydrophobic domain.

Assembly and budding of influenza virus

“…Furthermore, only the entire M1 (aa 1-252), which binds both RNA and the vRNP, causes transcription inhibition . Interaction between the M1 protein and RNA was demonstrated by using filter-binding assays and blotting procedures (Wakefield and Brownlee, 1989;Ye et al, 1989). However, such artificial interaction of M1 with single stranded RNA is non-specific.…”

Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication

“…In the influenza virus-infected cells, M1 regulates the nuclear export of vRNPs and prevents newly exported vRNP from re-entering the nucleus (Martin and Helenius, 1991). M1 may also be involved in the inhibition of viral transcription in the late stages of infection and regulation of the switch from replication to viral assembly and budding (Ye et al, 1989). The M1 protein harbours an N-terminal (N) domain (aa 2-67), a middle (M) domain (aa 91-158) and a C-terminal (C) domain (aa 165-252) (Sha and Luo, 1997).…”

The cytoplasmic tail of hantavirus Gn glycoprotein interacts with RNA

“…Hantaviruses do not possess an individual matrix protein and based on recent evidence, instead, the relatively large Gn-CT substitutes for this activity (Battisti et al, 2010;Hepojoki et al, 2010a;Huiskonen et al, 2010;Wang et al, 2010). The matrix protein of both family Bornaviridae (Neumann et al, 2009) and Orthomyxoviridae (Elster et al, 1997;Wakefield and Brownlee, 1989;Ye et al, 1989) viruses have been shown to bind, in addition to the viral core proteins, also the genomic RNA. Thus our results suggest by analogy the Gn-CT of hantaviruses to be equivalent of a matrix protein in the family Bunyaviridae.…”

“…Although the exact mechanism of genome incorporation in to virions is not known, it has been shown that the matrix protein M1 interacts with RNP (Baudin et al, 2001;Elster et al, 1997;Noton et al, 2007;Ye et al, 1999). The M1 protein harbors a ZF and a nuclear localization signal with RNA-binding abilities (Elster et al, 1997;Wakefield and Brownlee, 1989;Ye et al, 1999;Ye et al, 1989). Interestingly, the influenza M1 has been shown to repress endogenous viral transcription through its RNA-binding domains (Perez and Donis, 1998;Watanabe et al, 1996;Ye et al, 1989).…”

“…The M1 protein harbors a ZF and a nuclear localization signal with RNA-binding abilities (Elster et al, 1997;Wakefield and Brownlee, 1989;Ye et al, 1999;Ye et al, 1989). Interestingly, the influenza M1 has been shown to repress endogenous viral transcription through its RNA-binding domains (Perez and Donis, 1998;Watanabe et al, 1996;Ye et al, 1989). This activity could be biologically relevant in the late stages of infection where it would halt viral mRNA production and thereby trigger the packaging of viral genome eventually leading to the egress of virions.…”