Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

Abstract

Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

The replication of the human respiratory syncytial virus in a T cell line has multiple ineffective steps

“…Since HRSV uses the secretory pathway to deliver viral proteins to the assembly sites at the plasma membrane [1,15,17,19,23,24], we examined the presence of virus proteins along the main components of the secretory and endosomal pathways by immunofluorescence. Our results and previous literature have shown that HRSV F and N proteins partially co-localized with markers of the secretory pathway [1,23,24]. However, it was noteworthy that in contrast to HEp-2 cells, A3.01 cells did not display an evident accumulation of SNX2 at places where the HRSV N protein is.…”

“…HRSV N protein was also observed to co-localize partially with the Golgi [23]. Consistent with these findings, recently we have shown that in HEp-2 cells the M and N engage partially with secretory pathway and with the retromer complex [24]. Furthermore, HRSV P protein was found partially co-localizing with endosomal vesicles [25].…”

“…In epithelial cells, F protein, a transmembrane protein, follows the conventional anterograde pathway from ER through Golgi to the plasma membrane [36]. Also, the Golgi participates in the intracellular traffic of the HRSV N protein to the plasma membrane [23,24]. To examine whether the trafficking of HRSV F and N proteins in A3.01 lymphocytes is similar to the one in HEp-2 cells, immunofluorescence staining was done for a cis- and medial-Golgi marker, giantin, a trans-Golgi network marker, TGN46, and the viral proteins F and N. A partial co-localization of HRSV F protein with the Golgi markers was observed at 48 hours post-infection (Figure 7E-H).…”

“…After trafficking through the Golgi stacks, the transmembrane proteins often associate with endosomal pathways [33–35]. In addition, it is already known that some of the HRSV proteins follow the endosomal system to reach the plasma membrane [19,23,24,36]. To investigate whether the HRSV proteins also associate with endosomal machineries in A3.01 cells, immunofluorescence assays were performed for several markers of this pathway.…”

“…CD63 is a marker for late endosomes, while SNX2 is part of the cellular retromer complex, responsible for carrying cargoes from early endosomes to the trans-Golgi [37,38]. In addition, recently, it was found that SNX2 is recruited to HRSV N inclusion bodies and plays a role in the HRSV viral production in HEp-2 cells [24]. It was possible to see that, similarly to what happens in HEp-2 cells, the HRSV F protein partially co-localized with SNX2 and CD63 in A3.01 (figures 9I to L, and supplementary figures 2G to L).…”