Ana Asenjo

Interactions between cellular actin and human respiratory syncytial virus (HRSV)

Actin the main component of the cellular microfilament network, is present in human respiratory syncytial virus (HRSV) purified virions, as an internal component. This fact and the results of immunoprecipitation studies indicate that during HRSV infection in HEp-2 cells there are interactions between cellular actin and viral components, that can promote a transitory increase in the polymerization of synthetized actin, mainly of the beta isotype. This increased actin polymerization can be related with the formation of cytoplasmic extensions, that contain beta actin and viral particles observed in the HRSV infected HEp-2 cells. The formation of these structures may indicate that HRSV has developed an actin-based motility system similar to that described for other viral and bacterial systems.

Structural Phosphoprotein M2-1 of the Human Respiratory Syncytial Virus Is an RNA Binding Protein

ABSTRACT The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays. It is able to bind HRSV leader RNA specifically with cooperative kinetics, with an apparentKd of at least 90 nM. It also binds to long RNAs with no sequence specificity. The RNA binding domain has been located between amino acid residues 59 and 85, at the NH2terminus of the protein. This region contains the phosphorylatable amino acid residues threonine 56 and serine 58, whose modification decreases the binding capacity of M2-1 protein to long RNAs.

The bulk of the phosphorylation of human respiratory syncytial virus phosphoprotein is not essential but modulates viral RNA transcription and replication.

The ability of variants of the human respiratory syncytial virus (HRSV) phosphoprotein (P protein) to support RNA transcription and replication has been studied by using HRSV-based subgenomic replicons. The serine residues normally phosphorylated in P during HRSV infection have been replaced by other residues. The results indicate that the bulk of phosphorylation of P (98%) is not essential for viral RNA transcription or replication but that phosphorylation can modulate these processes.

Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization

Purified human respiratory syncytial virus (HRSV) P phosphoprotein from transfected HEp‐2 cells is able to oligomerize forming tetramers. The bulk of constitutive P protein phosphorylation (99.8%) (serine residues 116, 117, 119, 232 and 237) can be removed without affecting protein oligomerization. However, dephosphorylated P protein, produced in bacteria, is unable to oligomerize. This difference can be explained by a transient P protein phosphorylation, detected in HEp‐2 cells, that could be essential for P protein oligomerization.

Residues in human respiratory syncytial virus P protein that are essential for its activity on RNA viral synthesis.

Human respiratory syncytial virus (HRSV) P protein, 241 amino acid long, is a structural homotetrameric phosphoprotein. Viral transcription and replication processes are dependent on functional P protein interactions inside viral ribonucleoprotein complexes (RNPs). Binding capacity to RNPs proteins and transcription and replication complementation analyses, using inactive P protein variants, have identified residues essential for functional interactions with itself, L, N and M2-1 proteins. P protein may establish some of these interactions as monomer, but efficient viral transcription and replication requires P protein oligomerization through the central region of the molecule. A structurally stable three-dimensional model has been generated in silico for this region (residues 98-158). Our analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization. Additionally, the residues D136, S156, T160 and E179 appear to be essential for P protein activity on viral RNA synthesis and very high turnover phosphorylation at S143, T160 and T210 could regulate it. Thus, compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect.

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) beta and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.

Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells.

Phosphorylation of the human respiratory syncytial virus N protein provokes a decrease in viral RNA synthesis.

When HEp-2 cells are infected by human respiratory syncytial virus (HRSV) its N protein becomes phosphorylated at tyrosine (Y) Y38, in a strictly regulated way. To determine how this phosphorylation affects nucleocapsid (NC) template activity during viral RNA synthesis, N protein variants were analysed in which Y38 and nearby Y residues were substituted by phenylalanine (F; Y23F, Y38F and Y69F) or aspartic acid (D; Y23D and Y38D). While the capacity of these proteins to form the NC and to interact with the P protein was maintained, their NC template activity was altered affecting distinctly viral transcription and replication of HRSV based minigenomes. Thus, Y38 phosphorylation of the HRSV N protein modulates NC template activity probably by altering the interactions of the monomeric components of the NC.