Sorting of the Respiratory Syncytial Virus Matrix Protein into Detergent-Resistant Structures Is Dependent on Cell-Surface Expression of the Glycoproteins

Abstract

The interaction of the respiratory syncytial virus (RSV) Matrix (M) protein with the plasma membrane was investigated using polyclonal and monoclonal antisera raised against recombinant M expressed in bacteria. M bound mainly to the plasma membrane, although a significant proportion bound to internal membranes. However, no localisation of M with the Golgi was observed, suggesting that transport of M to the plasma membrane was independent of the transport mechanism for the viral glycoproteins. Expression from a recombinant baculovirus demonstrated the ability of M to bind membranes in the absence of viral glycoprotein expression. When cell-surface expression of the viral glycoproteins was prevented using Brefeldin A, M was still found in association with the plasma membrane, but the characteristics of M's membrane-binding ability were different to that found in untreated infected cells. In the presence of normal glycoprotein expression, M was sorted into lipid rafts and, in addition, formed structures that could only be disrupted by treatment with high salt buffers, a feature suggesting an interaction with the cytoskeleton or the formation of strong intramolecular associations. Brefeldin A prevented M from being sorted into lipid rafts or from forming strong intramolecular associations. Brefeldin A also affected the stability of M bound to the plasma membrane, as M was more readily dissociated in the presence of the inhibitor. Coexpression of M and F resulted in the incorporation of M into lipid rafts but did not cause the formation of the strong intramolecular bonds, suggesting that additional factors are required for this phenomena.

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

“…M was shown previously to associate with viral RNPs through interactions with the M2-1 protein and also has an RNA-binding capacity (35,52). M binds both plasma and internal cellular membranes and was reported to interact with the G protein cytoplasmic tail (CT) (20,27). Several of these observations agree with a predicted role for M in the late stages of virion production.…”

“…HRSVinduced surface filaments have been suggested to be the equivalent of virions (27,49,53,60), and the loss of viral filaments typically results in the loss of the majority of viral infectivity (7,42,49,53). An exception to the above-mentioned data was a study in which wt HRSV-infected HEp-2 cells were treated with RhoA inhibitors (22).…”

“…F also appears to play a role in viral attachment, and nucleolin was recently identified as a cellular receptor for the F protein (57). M is a nonglycosylated phosphorylated protein of 256 amino acids and a structural component of the HRSV virion (19,27,52). M is thought, in part by analogy to the role of matrix proteins in other virus systems, to play a key role in virion assembly by inhibiting viral transcription and by forming a bridge between the viral ribonucleoprotein (RNP) and envelope (13,21,25,27,54,56).…”

“…Several reports have implicated cytoskeletal elements in the processes that lead to virus egress, in particular microtubules, myosin V, actin, and actin-regulatory proteins such as profilin and RhoA (30,32,59). In addition, a role for an actin/myosinbased motility system in HRSV exit has been proposed (6,53,59), and there is strong evidence for the involvement of lipid rafts (8,9,22,27,31,36,49,50,53).…”

“…Studies of host cell infection by HRSV in the absence of the SH, G, and F proteins pointed to M as an important determinant of virion release (2,3). The roles of M in the viral assembly process likely include a function in bringing together the RNP and the viral envelope, since an M-containing sheath was revealed when the lipid membrane was removed from HRSV-induced surface filaments (1,27). In HRSV-infected cells, the M protein is first detected in the cytoplasm and nucleus (19).…”