In Vitro Dissection of the Membrane and RNP Binding Activities of Influenza Virus M1 Protein

Abstract

Spontaneous proteolysis of influenza virus M1 protein during crystallisation has defined an N-terminal domain of amino acids 1--164. Full-length M1, the N-terminal domain, and the C-terminal part of M1 (residues 165--252) were produced in Escherichia coli. In vitro tests showed that only full-length M1 and its N-terminal domain bind to negatively charged liposomes and that only full-length M1 and its C-terminal part bind to RNP. However, only full-length M1 had transcription inhibition activity. Several independent experimental approaches indicate that in vitro transcription inhibition occurs through polymerisation/aggregation of M1 onto RNP, or of M1 onto M1 already bound to RNP, rather than by binding to a specific active site on the nucleoprotein or the polymerase. The structure/function of influenza virus M1 will be compared with that of the Ebola virus matrix protein, VP40.

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association

“…However, the mechanism of M1-RNP interaction remains poorly understood. FLUA-M1 has been shown to bind NP (45), RNA (34,36), as well as RNP (4,35,46). In particular, Baudin et al demonstrated that it is the FLUA-M1 C domain, instead of the N domain, that is required for RNP binding (4).…”

“…FLUA-M1 has been shown to bind NP (45), RNA (34,36), as well as RNP (4,35,46). In particular, Baudin et al demonstrated that it is the FLUA-M1 C domain, instead of the N domain, that is required for RNP binding (4). In a more recent study, it was shown that FLUA-M1 is SUMOylated at K242 from the C domain, and that the SUMOylation of M1 is required for the interaction between M1 and viral RNP (46).…”

“…As infection proceeds, newly synthesized M1 enters the nucleus to mediate the nuclear export of nascent vRNPs. A "daisy-chain" complex is formed with the vRNP binding to the C-terminal domain of M1 (4) and the N-terminal domain of M1 interacting with the nuclear export protein (NEP), also called NS2. Through its nuclear export signal (NES), NEP is specifically recognized by the cellular exportin Crm1, which then facilitates the transport of vRNPs across the nuclear membrane to the cytoplasm in a RanGTP-dependent manner (5).…”

“…Through its nuclear export signal (NES), NEP is specifically recognized by the cellular exportin Crm1, which then facilitates the transport of vRNPs across the nuclear membrane to the cytoplasm in a RanGTP-dependent manner (5). It has been shown that M1 binding to vRNP in the nucleus is able to block mRNA transcription (4,6). In the cytosol, M1 interacts with the cytoplasmic tails of the glycoproteins HA and NA.…”

NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome

“…5a and b). Previous studies have shown that expression of the M1 protein can repress transcriptional activity of RNPs (Baudin et al, 2001;Elster et al, 1997;Watanabe et al, 1996;Ye et al, 1987Ye et al, , 1989, which could be an explanation for the lower RNA levels observed.…”