Respiratory Syncytial Virus Matrix Protein Research Articles

Central role of the respiratory syncytial virus matrix protein in infection

Respiratory syncytial virus is the major respiratory pathogen of infants and children worldwide, with no effective treatment or vaccine available. Steady progress has been made in understanding the respiratory syncytial virus life cycle and the consequences of infection, but many areas of respiratory syncytial virus biology remain poorly understood, including the role of subcellular localisation of respiratory syncytial virus gene products such as the matrix protein in the infected host cell. The matrix protein plays a central role in viral assembly and, intriguingly, has been observed to traffic into and out of the nucleus at specific times during the respiratory syncytial virus infectious cycle. Further, the matrix protein has been shown to be able to inhibit transcription, which may be a key to respiratory syncytial virus pathogenesis. This review will focus on the role of the matrix protein in respiratory syncytial virus infection and what is known of its nucleocytoplasmic trafficking, the understanding of which may lead to new therapeutic approaches to combat respiratory syncytial virus, and/or vaccine development.

Nuclear Import of the Respiratory Syncytial Virus Matrix Protein Is Mediated By Importin β1 Independent of Importin α

The matrix (M) protein of respiratory syncytial virus (RSV) plays an important role in virus assembly through specific interactions with RSV nucleocapsids and envelope glycoproteins in the cytoplasm as well as with the host cell membrane. We have previously shown that M localizes to the nucleus of infected cells at an early stage in the RSV infection cycle, where it may be instrumental in inhibiting host cell processes. The present study uses transient expression of M as well as a truncated green fluorescent protein (GFP) fusion derivative to show for the first time that M is able to localize in the nucleus in the absence of other RSV gene products, through the action of amino acids 110-183, encompassing the nucleic acid binding regions of the protein, that are sufficient to target GFP to the nucleus. Using native PAGE, ELISA-based binding assays, a novel Alphascreen assay, and an in vitro nuclear transport assay, we show that M is recognized directly by the importin beta1 nuclear import receptor, which mediates its nuclear import in concert with the guanine nucleotide-binding protein Ran. Retention of M in the nucleus through binding to nuclear components, probably mediated by the putative zinc finger domain of M, also contributes to M nuclear accumulation. This is the first report of the importin binding and nuclear import properties of a gene product from a negative sense RNA virus, with implications for the function of RSV M and possibly other viral M proteins in the nucleus of infected cells.

Interaction between the respiratory syncytial virus G glycoprotein cytoplasmic domain and the matrix protein

Paramyxovirus assembly at the cell membrane requires the movement of viral components to budding sites and envelopment of nucleocapsids by cellular membranes containing viral glycoproteins, facilitated by interactions with the matrix protein. The specific protein interactions during assembly of respiratory syncytial virus (RSV) are unknown. Here, the postulated interaction between the RSV matrix protein (M) and G glycoprotein (G) was investigated. Partial co-localization of M with G was demonstrated, but not with a truncated variant lacking the cytoplasmic domain and one-third of the transmembrane domain, in cells infected with recombinant RSV or transfected to express G and M. A series of G mutants was constructed with progressively truncated or modified cytoplasmic domains. Data from co-expression in cells and a cell-free binding assay showed that the N-terminal aa 2-6 of G play a key role in G-M interaction, with serine at position 2 and aspartate at position 6 playing key roles.

Sequence and structure relatedness of matrix protein of human respiratory syncytial virus with matrix proteins of other negative-sense RNA viruses

Matrix proteins of viruses within the order Mononegavirales have similar functions and play important roles in virus assembly. Protein sequence alignment, phylogenetic tree derivation, hydropathy profiles and secondary structure prediction were performed on selected matrix protein sequences, using human respiratory syncytial virus matrix protein as the reference. No general conservation of primary, secondary or tertiary structure was found, except for a broad similarity in the hydropathy pattern correlating with the fact that all the proteins studied are membrane-associated. Interestingly, the matrix proteins of Ebola virus and human respiratory syncytial virus shared secondary structure homology.

Human respiratory syncytial virus matrix protein is an RNA-binding protein: binding properties, location and identity of the RNA contact residues.

The human respiratory syncytial virus (HRSV) matrix (M) protein is a structural internal membrane protein. Here we have shown that, like its orthomyxovirus and rhabdovirus counterparts, it has RNA-binding capacity, as determined by retardation of (32)P-labelled riboprobes in gel electrophoresis, cross-linking with UV light and Northern-Western assays. Its binding to RNA was neither sequence-specific nor showed a length requirement, although it had cooperative kinetics with a K(d) of 25 nM and probably two different types of RNA-binding sites. After preparative cross-linking of (32)P-labelled riboprobes with purified, renatured HRSV Long strain M protein (256 residues), the residues in contact with RNA were located between amino acids 120 and 170, in the central part of the molecule. Lysine (positions 121, 130, 156 and 157) and arginine (position 170) residues located within this region and conserved among pneumovirus M proteins of different origins were found to be essential for RNA contact. M protein expression did not affect the replication and transcription of HRSV RNA analogues in vivo (except when expressed in large amounts), in contrast to the in vitro transcription inhibition described previously. In addition, M protein was found to aggregate into high-molecular-mass oligomers, both in the presence and absence of its RNA-binding activity. The formation of these structures has been related in other viruses to either viral or host-cell RNA metabolism.

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

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.

Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells.

Little is known about the functions of the matrix (M) protein of respiratory syncytial virus (RSV). By analogy with other negative-strand RNA viruses, the M protein should inhibit the viral polymerase prior to packaging and facilitate virion assembly. In this study, localization of the RSV M protein in infected cells and its association with the RSV nucleocapsid complex was investigated. RSV-infected cells were shown to contain characteristic cytoplasmic inclusions. Further analysis showed that these inclusions were localization sites of the M protein as well as the N, P, L and M2-1 proteins described previously. The M protein co-purified with viral ribonucleoproteins (RNPs) from RSV-infected cells. The transcriptase activity of purified RNPs was enhanced by treatment with antibodies to the M protein in a dose-dependent manner. These data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription.

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.