Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding

Abstract

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.The final step in the lytic cycle of enveloped viruses involves the budding of the newly formed particles from cellular membranes. Previous to this step, all viral structural components should have been transported, either individually or as preassembled complexes, to the cellular membrane, where viral proteins will drive the budding process.A number of studies have focused on the assembly and budding processes of viruses (arena-, alpha-, rhabdo-, paramyxo-, orthomyxo-, and retroviruses) that obtain their envelope from the plasma membrane (reviewed in references 2, 13, and 19). For the alphavirus Semliki Forest virus, it has been established that virus budding is strictly dependent on interactions between the transmembrane spike protein and the internal nucleocapsid (46). In retroviruses, however, interactions between the cytoplasmic tail of external virus proteins (Env) and the internal virus components (Gag polyprotein) are not a prerequisite for virus budding since expression of the Gag protein alone is sufficient to drive budding of virus-like particles (VLPs) (7,14). A different mechanism, which directs the assembly and release of coronavirus particles, which assemble at intracellular membranes, has been described (47). In this case, expression of viral membrane proteins alone is sufficient to drive the assembly and budding of VLPs (47).It is widely accepted that the matrix protein plays a pivotal role as an assembly organizer for RNA viruses containing a single negative-strand genomic RNA molecule (such as rhabdo-and paramyxoviruses) (reviewed in reference 25). In fact, rabies and measles viruses modified by reverse genetics technology to lack the matrix gene grow poorly, and the released matrix-less particles show drastically altered morphologies (3, 31). Moreover, it has been shown that the M1 proteins of vesicular stomatits virus (VSV) and human parainfluenza virus type 1 have intrinsic budding activity when expressed alone (5, 22, 26), an observation ...

Identification of Cellular Interaction Partners of the Influenza Virus Ribonucleoprotein Complex and Polymerase Complex Using Proteomic-Based Approaches

“…21 The SEQUEST software (Thermo Finnigan) was used to search the NCBI non-redundant protein data base (www.ncbi.nlm.nih.gov, downloaded 06/13/2003). Data files were created by the SEQUEST software for every MS/MS scan with a total ion count of at least 5×10 4 , minimal peak count of 35, and a precursor ion mass in the range of 300-2000 m/z. Data were searched against the data base restricted to tryptic peptides without modifications (except for carboxyamidomethylated cysteines 57.0513 and oxidized methionines 15.9994) allowing a parent mass error tolerance of 2 Da and daughter ion error tolerance of 0.8 Da.…”

“…This assembly is apparently mediated by the M1 protein which has also been hypothesized to be the driving force in the budding process. 4 The various functions of vRNP during the viral life cycle suggest that many cellular factors associate to this complex, fulfilling different functions. In addition, the interaction of vRNP components with cellular proteins may also determine the host specificity of influenza virus.…”

Formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase

“…M1 affects virus assembly by interacting with the core viral ribonucleocapsid (vRNP) and cytoplasmic tail of transmembrane proteins, forming a bridge between the two layers, as well as recruiting the internal viral proteins and viral RNA to the plasma membrane in a cooperative manner (Noda et al, 2006). In addition, M1 interacts with the lipid bilayer producing an outward bending of the membrane, and this has been postulated to be the major driving force of influenza budding, as cells expressing M1 protein alone produce virus-like particles (VLPs) (Gó mez-Puertas et al, 2000;Latham & Galaeza, 2001). …”

Characterization of the 1918 “Spanish” Influenza Virus Matrix Gene Segment

“…M1 binds RNA (41,49,57,59), vRNPs (34,38,41,57), and lipids (7,15); dimerizes with other M1 molecules (16,41); and interacts with both the HA and neuraminidase proteins (1,11). It is also involved in export to the cytoplasmic membrane, virus assembly, and budding (12,19,31).…”