# Encapsidation of Staufen-2 Enhances Infectivity of HIV-1
## 3 Results
### Multiple Pathways Mediate Staufen-2 Incorporation into Virions  
#### 3.3.1 Staufen-2 Interacts with Staufen-1  
Staufen-1 and Staufen-2 have been reported to interact with each other in vitro [22]. To investigate if these two proteins interact with each other during HIV-1 propagation, we performed immunoprecipitation assays in HEK293T cells transfected with either pcDNA3 or pcDNA3 RSV Stau-1 HA (Staufen-1 with HA tag) using an anti-HA affinity matrix. The pull-down product was probed with an anti-Staufen-2 antibody. Figure 4A shows that Staufen-2 is detectable in Staufen-1 immuno-precipitate independent of treatment with RNase A, supporting an RNA-independent interaction of Staufen1-HA with Staufen-2 (Figure 4A). In addition, we performed a reverse IP, wherein we pulled FLAG-tagged Staufen-2 to investigate if it coprecipitates with HA-tagged Staufen-1 (Supplementary Figure S3A).  
Figure 3: Staufen-2 interacts with HIV-1 Gag: (**A**) HEK293T cells were co-transfected with either pkGST or pkGST-HIV-1 Gag and Staufen-2 FLAG or Staufen-1 FLAG (positive control) constructs. The overexpression of proteins was confirmed by Western analyses (input). After 48 h of transfection, total cell lysates were prepared, and a GST pull-down assay was performed, followed by Western analyses with anti-FLAG (Staufen-2 or Staufen-1) and anti-GST (HIV-1 Gag GST or GST alone) antibodies. (**B**) GST pull-down assay was performed with or without RNase A treatment of the total cell lysates and probed with anti- FLAG (Staufen-2 or Staufen-1) and anti-GST (HIV-1 Gag GST or GST alone) antibodies. GAPDH served as a loading control in both A and B. (**C**) Staufen-2 FLAG, or Staufen-1 FLAG and HIV-1 Gag-EGFP were localized by HIV-1 Gag (EGFP) or Staufen-2 and Staufen-1 (FLAG-TR red). Nuclei are stained by DAPI (in blue). The co-localized complexes are seen as yellow rings in the merged panel. (**D**) The RNF complexes (with or without RNase A treatment) were separated based on their molecular size and density. Each fraction was checked for the presence of the transiently expressed Staufen-2 FLAG and HIV-1 Gag by Western analyses. Ribosomal protein S6 was used as a control.  
The interaction between Staufen-1 and Staufen-2 was also confirmed by confocal microscopy, wherein Staufen-1 and Staufen-2 co-localized in the cytoplasm (Figure 4B and Supplementary Figure S3B). Upon investigating the RNPs extracted by sucrose step-gradient centrifugation from HEK293T cells over-expressing Staufen-2, we observed that endogenous Staufen-1 and overexpressed Staufen-2 FLAG co-associated in the same RNP fractions, which was partially affected by RNase A treatment, confirming their interactions under physiological conditions (Figure 4C). With the co-localization in the same RNPs, we additionally observed that both Staufen-1 and Staufen-2 were found in viral lysates from the cells transiently overexpressing these proteins, indicating the possibility of encapsidation of these proteins together in emerging virions (Supplementary Figure S4A), although individual proteins getting differentially packaged within the viral population cannot be ruled out.