Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein

Abstract

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.Influenza viruses, enveloped RNA viruses containing singlestranded, segmented RNA of negative polarity, assemble and bud from the plasma membrane of virus-infected cells into the outside environment. Complete virions are usually not observed inside the cell in the productive infectious cycle. Furthermore, in polarized epithelial cells, influenza viruses bud asymmetrically, i.e., predominantly from the apical plasma membrane (30). For virus budding to occur, two processes are obligatory (27). Firstly, all viral structural components, namely, the matrix protein (M1), the viral nucleocapsid (viral ribonucleoprotein [vRNP]) containing vRNA, nucleoprotein (NP), polymerase proteins (PB1, PB2, and PA), and NS2 (NEP) as well as the viral envelope containing the host lipids and three transmembrane proteins (hemagglutinin [HA], neuraminidase [NA], and M2) must be transported and targeted either individually or as complex subviral components to the assembly site at the plasma membrane. Secondly, these viral proteins and/or subviral components must interact with each other to initiate the budding processes leading to morphogenesis of virus particles and release of virions.Influenza virus M1, the most abundant protein in the virus particle,...

Improvement of H5N1 influenza vaccine viruses: Influence of internal gene segments of avian and human origin on production and hemagglutinin content

“…To this purpose a set of H5N1-PR8 reassortant influenza viruses with M or PB1 segments derived from avian or human strains were generated and investigated regarding replication in embryonated chicken eggs and HA content. We chose this strategy as the viral M1 protein interacts with surface glycoproteins and contributes to virus assembly [22], whereas the cytoplasmic tail of the M2 protein is required for efficient genome packaging [24] and also contributes to virus assembly [35]. The PB1 gene was included as it was inherited in some circumstances from a seasonal WT isolate or the pandemic H1N1-2009 virus to the corresponding vaccine strains during classical reassortment.…”

“…The M1 protein is located underneath the particle membrane [21] and is described to interact with the virus surface glycoproteins as well as with the vRNPs in the virus particle [22] and [23]. This interaction is thought to be essential for assembly and budding of virus particles [22].…”

“…The M1 protein is located underneath the particle membrane [21] and is described to interact with the virus surface glycoproteins as well as with the vRNPs in the virus particle [22] and [23]. This interaction is thought to be essential for assembly and budding of virus particles [22]. Moreover, the cytoplasmic tail of the M2 protein was found to be involved in the production of new virus particles and the efficient packaging of virus genomes [24] and [25].…”

The packaging signal of influenza viral RNA molecules

“…RT-PCR analysis for the presence of vRNA and/or cRNA in the cytoplasm of cells infected by pHL1354 (A) or pHL1844 (B) recombinant viruses at 5 h postinfection+ Reverse transcription for CAT-specific vRNAs and cRNAs followed by PCR amplification were done similarly to Figure 4 with DNAse pretreated cytoplasmic and nuclear fractions of MDCK cells infected by pHL1354 or pHL1844 recombinant viruses, respectively; at the second step of viral passage+ A control PCR reaction (lane C), which was not preceded by an RT reaction, was done in each case to prove the absence of plasmid DNA in the cytoplasmic RNA preparation+ The purity of the cytoplasmic and nuclear fractions was controlled by canine p53 hnRNA/mRNA RT-PCR across intron 7 with expected fragments of 289 bp (mRNA) and 551 bp (hnRNA)+ Expected PCR products for pHL1354 CAT-cRNA (668 bp) (A) and for pHL1844 CAT-vRNA (855 bp) (B) have been indicated at the left margin, and the p53 RT-PCR product sizes are marked at the right margin+ For sizes of marker bands (lanes M) see Figure 4, right margin+ (Murti et al+, 1992)+ These interactions have not so far been differentiated with regard to the RNP constituents, NP and polymerase+ Due to the demonstrated requirement of a (59-bulged) vRNA promoter structure for nuclear export, we propose an M1 interaction with RNA-polymerase rather than NP, in the respective vRNA-bound conformation, with or without assistance by other components as listed above+ This conclusion is also supported by the published failure to demonstrate an NP-M1 interaction in vivo in the absence of polymerase (Zhao et al+, 1998), whereas various RNA polymerase activities, in particular initiation reactions, have been shown to be inhibited by M1 addition, both in vivo (Watanabe et al+, 1996) and in vitro (Ye et al+, 1987)+ In a potential chain of protein interactions leading to vRNP nuclear export, viral NS2 is likely to occupy a more downstream position because of its specific interaction with cellular CRM1, which in turn interacts with the standard cellular machinery such as Ran-GTP, and ultimately with components of the nuclear pore complex (Neumann et al+, 2000)+ However, NS2's mode of "upstream" interaction with vRNP, M1, and/or other factors is not known at present+ Incorporation into the budding progeny virion of several cytoplasmic vRNPs in association with M1 and/or cellular transport factors has to be brought about through interaction with other partners present in that new compartment at the plasma membrane+ Likely candidates in this regard are the negatively charged phospholipid membrane itself (Ruigrok et al+, 2000), or interactions of M1 with the cytoplasmic tails of the glycoproteins HA and NA (Mitnaul et al+, 1996;Zhou et al+, 1998;Ali et al+, 2000) as well as M2+ Such interactions are known to bring about conformational changes in M1 during budding, whereas their absence results in aberrant influenza particle shapes (Jin et al+, 1997)+…”

Flu channel drug resistance: a tale of two sites

“…Therefore, the membrane patch that is destined to bud out from the host cell must contain specific sites for recruiting the matrix protein and RNP complexes. AM1 can directly bind to the viral membrane via electrostatic interaction (Ali et al, 2000). But membrane alone is clearly insufficient to warrant specificity, because charged membranes of other organelles are present in the cell.…”