# Encapsidation of Staufen-2 Enhances Infectivity of HIV-1
## 3 Results
### Multiple Pathways Mediate Staufen-2 Incorporation into Virions
#### 3.3.2 Staufen-2 Interacts with APOBEC3G  
Given the similarity of APOBEC3G (A3G) and Staufen-2 in binding to HIV-1 Gag and Staufen-1, we investigated if Staufen-2 and A3G can also interact with each other. Similar to the experiments performed to establish interactions between Staufen-2 and Staufen-1, immunoprecipitation assays using anti-HA affinity matrix in HEK293T cells expressing HA-tagged A3G and FLAG-tagged Staufen-2 were performed with FLAG-tagged vector as a negative control. Indeed, we detected Staufen-2 in A3G precipitates that were not affected by RNase A treatment or HIV-1 proviral DNA co-expression (Figure 5A). However, RNAse A treatment disrupted the A3G oligomerization, as described before [59] (Figure 5B). Staufen-2 interaction with A3G was also observed through confocal microscopy of co-expressing cells (Figure 5C). Interestingly, Staufen-2 co-localized within the RNPs  
Figure 4: Staufen-2–Staufen-1 interaction is RNA-independent: (**A**) HEK293T cells were transfected with vector alone or with Staufen-1 construct. After 48 h of transfection, total cell lysates were prepared, followed by IP with anti-HA affinity matrix was performed without or with RNase A treatment. The expression of Staufen-1 (anti-HA) and Staufen-2 (anti-Stau-2) were confirmed by Western analyses with GAPDH as a loading control. (**B**) Staufen-2 FLAG (red) or Staufen-1 HA (green) were localized by confocal microscopy. Nuclei were stained by DAPI (in blue). The co-localized complexes are seen as yellow rings in the merged panel. (**C**) The RNP complexes, either with or without RNase A treatment, were separated based on their molecular size and density. Each fraction was monitored for Staufen-2 FLAG and Staufen-1 by Western analyses with ribosomal protein S6 as a control for the RNP complex. carrying A3G, the association of which was found to be affected by RNase A treatment (Figure 5D). Fractions isolated by sucrose density gradient centrifugation of co-expressing cells suggested that a part of the Staufen-2 interaction with A3G is mediated by RNA, similar to, as reported earlier for Staufen-1 and A3G interaction [60, 61]. RNase A treatment of RNP complexes majorly affects A3G and Staufen-1, but not Staufen-2 (Figures 4C and 5D). This probably suggests an RNA-independent multimerization of Staufen-2 in cells, as well as a possibility of its co-association with other proteins in the RNP complex. Fate of the Viral Population with Encapsidated Staufen-2 Alone or with Its Partners Staufen-1 or A3G  
Having confirmed that Staufen-2 can be encapsidated alone via interacting with HIV-1 Gag, we also observed Staufen-2 and Staufen-1 or A3G in the viral particles (speculatively indicating the chance of co-packaging of these proteins) (Supplementary Figure S4A,B). Figure 5: Staufen-2 interacts with antiviral protein A3G: (**A**) HEK293T cells were co-transfected with either vector alone or A3G HA and Staufen-2 FLAG with or without HIV-1 proviral construct. After confirming the expression of individual proteins by Western analyses (input), total cell lysates were prepared 48 h post-transfection, followed by IP with anti-HA affinity matrix without or with RNase A treatment and probed with anti-FLAG (Staufen-2) and anti-HA (A3G) antibodies. (**B**) HEK293T cells were co-transfected with either vector (HA) or A3G HA-tagged and A3 GHis-tagged constructs. Total cell lysates were prepared, either with or without RNase A treatment, followed by immunoprecipitation with anti-HA beads and analyzed by Western blotting using anti-His antibodies. GAPDH served as a loading control. (**C**) Staufen-2 FLAG and A3G HA were localized by A3G HA (AF488 Green) or Staufen-2 FLAG (AF594 red). Nuclei were stained by DAPI (in blue). The co-localized complexes are seen as yellow rings in the merged panel. (**D**) The RNP complexes (with or without RNase A treatment) were separated based on their molecular size and density. Each fraction was monitored for the presence of Staufen-2 FLAG and A3G HA by Western analyses.