Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

Abstract

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95% of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.

Virus Infection and Lipid Rafts

“…The oligomerization of the viral matrix protein VP40, which plays a critical role in Ebola virus assembly and budding, is regulated in the plasma membrane by association with lipid rafts. 27,54) Paramyxoviruses such as respiratory syncytial virus (RSV), [55][56][57] Sendai virus, 58) and measles virus (MV) 52,59) use lipid rafts for assembly and budding. RSV polymerase complex associates with lipid rafts in virus-infected cells.…”

Differential Regulation of GM1 and Asialo-GM1 Expression by T Cells and Natural Killer (NK) Cells in Respiratory Syncytial Virus Infection

“…At the level of the virus life cycle, RSV replication machinery colocalizes with lipid rafts, and virions are selectively assembled at and likely bud from lipid rafts (50)(51)(52)(53)(54). GM1 is incorporated into the RSV viral envelope (55).…”

Role of Membrane Rafts in Viral Infection

“…Involvement of membrane rafts in viral RNA synthesis of enveloped viruses has been reported for HCV [143][144][145][146], respiratory syncytial virus (RSV; Paramyxoviridae) [147,148], DEN [149], and JEV [149]. Association of HCV nonstructural (NS) proteins with cholesterol-enriched membrane rafts in the Golgi-derived membrane forms the replication site of HCV RNA synthesis and protects it from RNase and protease [143,144].…”

“…The M protein is located between viral envelope and the RNP. The association of RSV RNP with membrane rafts leads to efficient RNA polymerase activity that may be required for interaction with the cellular factor HSP70, which is one of the virus receptor candidates, via membrane rafts in an ATP-dependent manner [147,148].…”

“…The role of membrane rafts in the assembly and budding of enveloped viruses has been investigated for influenza virus [59,63,[150][151][152][153][154][155][156][157][158][159][160], HIV-1 [63,[161][162][163][164][165][166][167][168][169][170][171][172][173], HTLV-1 [174], measles virus (Paramyxoviridae) [63,175,176], Sendai virus [Paramyxoviridae] [177,178], Newcastle disease virus (NDV; Paramyxoviridae) [179,180], RSV [147,148,[181][182][183][184], HSV [185,186], murine cytomegalovirus (MCMV; Herpesviridae) [187], Ebola virus [70,188], Marburg virus [70], and vesicular stomatitis virus (VSV; Rhabdoviridae) …”