Date Published: April 11, 2013
Publisher: Public Library of Science
Author(s): Magdalena Anna Krzyzaniak, Michael Thomas Zumstein, Juan Atilio Gerez, Paola Picotti, Ari Helenius, Andrew Pekosz.
Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na+/H+ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.
Human respiratory syncytial virus (RSV) belongs to the Paramyxoviridae, a family of enveloped viruses with a negative-stranded RNA genome. It is a ubiquitous human pathogen that causes severe respiratory tract infections affecting mainly children and the elderly worldwide. Despite ongoing efforts, there are no available vaccines or treatments except passive immunoprophylaxis . A better understanding of virus/host cell interactions is critical for the development of new therapeutic strategies.
Paramyxoviruses are generally thought to infect cells by fusing directly with the PM , . That paramyxovirus particles can also be endocytosed is, however, also clear. This has most recently been documented for Sendai, Nipah, RSV, Newcastle Disease viruses and for a lentivirus vector pseudotyped with measles virus glycoproteins , , , , , . Which of the two pathways – fusion at PM or fusion after endocytosis – leads to infection is not clear.