# Encapsidation of Staufen-2 Enhances Infectivity of HIV-1
## 3 Results  
### Staufen-2 Is Encapsidated into HIV-1 Virions, Enhancing Their Infectivity  
To investigate if Staufen-2 is packaged into HIV-1 virions and modulates viral infectivity, we generated HEK293T cells that stably expressed Staufen-2 (isoform 59 kDa) with a FLAG tag (Figure 1A). The Staufen-2 stably-expressing cells, as well as vector-integrated (control) cells, were transfected with an expression plasmid for the HIV-1 luciferase reporter virus (NL-LucR\({}^{-}\) E\({}^{-}\) along with pMDG.VSV-G) [46]. To assess the level of Staufen-2 expression in cell lysates and the presence of Staufen-2 in viral particles, Western blot analyses were performed 48 h post-transfection (Figure 1B). Staufen-2 was detected in both cell and viral lysates prepared from stably expressing cells and was absent in the control cells (Figure 1B). In addition, we isolated HIV-1 viral cores by sucrose density gradient centrifugation and evaluated the presence of Staufen-2 in these viral compartments (Supplementary Figure S1). Next, we evaluated the impact of Staufen-2 packaging on viral infectivity using a luciferase-based infectivity assay. HIV-1 NL-LucR\({}^{-}\) E\({}^{-}\) viral particles were produced as VSV-G pseudotypes in HEK293T vector and Staufen-2 stable cell lines. An equal amount of RT-activity normalized viral particles were used to infect HEK293T cells, and the luciferase activity was determined 48 h later. Virions of Staufen-2 cells had a 2.5-fold enhancement of their infectivity, suggesting a positive influence of Staufen-2 on viral infectivity (Figure 1C). To further characterize the role of Staufen-2 in HIV-1 infectivity, we generated HEK293T-Staufen-2 knockout (KO) cells (Supplementary Figure S2A,B) as well as Staufen-1 KO cells as a control (Supplementary Figure S2C,D). As a lower viral infectivity phenotype was reported in the Staufen-1 KO cell [12; 35], we performed a side-by-side comparison using Staufen-2 and Staufen-1 KO cells infected with HIV-1 Luc reporter viruses. It was observed that while both reduced viral infectivity, Staufen-2 KO background reduced the viral infectivity by 55%, while Staufen-1 KO background reduced the viral infectivity by 33%. Thus, the reduction in the viral infectivity was marginally more in Staufen-2 KO than Staufen-1 KO (Figure 1D). HEK293T cells were then infected with normalized virionsproduced from WT and KO cell lines. Virions generated from Staufen-1 KO and Staufen-2 KO cells showed a 25% and 34% reduction in infectivity, respectively, compared to virions from WT cells (Figure 1E). Similar results were observed in an infectivity assay using TZM-bl reporter cells infected with replication-competent NL4.3 HIV-1 virions containing Staufen proteins (Figure 2). Together, the data indicate a possible impact of the packaged Staufen-2 on viral infectivity. Figure 1: Staufen-2 is incorporated into HIV-1, enhancing viral infectivity: (**A**) Staufen-2 stable cell line was generated by transfection of HEK293T cells with pLYX-IRES-PURO Vector alone (vector cells) or pLYX-IRES-PURO-Staufen-2 FLAG and selected using puromycin. The stable overexpression of Staufen-2 FLAG proteins was confirmed by Western analyses using anti-FLAG antibodies. (**B**) Vector and Staufen-2 stable cell lines were transfected with NL-Luc R\({}^{-}\) E\({}^{-}\). After 48 h of transfection, the total cell and viral lysates were prepared. Western analyses were carried out with anti-FLAG (Staufen-2) and anti-p24 antibodies (HIV-1 capsid protein) in the cell viral lysate. GAPDH served as a loading control in both A and B. (**C**) NL-Luc R\({}^{-}\) E\({}^{-}\) viral particles were produced in vector and Staufen-2 stable cell lines. An equal amount of RT-activity normalized viral particles were used for the luciferase-based infectivity assay in HEK293T cells. Viral infectivity was quantified relative to the virus lacking Staufen-2. The presented values represent mean \(\pm\) standard deviations (error bars) for three independent experiments. * \(p\)-value \(\leq\) 0.05 was considered significant.