# Encapsidation of Staufen-2 Enhances Infectivity of HIV-1
## 4 Discussion  
During the assembly and budding of HIV-1, several host factors remain associated with the newly emerging viruses [62; 63; 64]. These virus-associated or encapsidated host proteins may influence the infectivity of the virus population and decide the fate of disease progression [65; 66; 67]. Since Staufen-1, one such protein, is known to be packaged in several RNA viruses and acts as a positive effector for viral infectivity [12; 14; 35], here we comprehensively analyzed the viral packaging efficiency of Staufen-2, especially in HIV-1 for the first time. Herein, we report Staufen-2 expressed in virus producer cells is efficiently incorporated into HIV-1 particles potentially via Gag interaction and promotes their infectivity. This expands our knowledge of different Staufen proteins' function in HIV-1 replication. Our previous study identified Staufen-2 as a host factor that is upregulated in Sup-T1 and likely in other cells during HIV-1 infection. It can enhance virus production by directly interacting with Rev and facilitating its RNA export activity [40]. Here, we essentially investigated its protein binding features and confirmed that HIV-1 Gag, Staufen-1, and A3G are indeed some interacting partners. Figure 6: Impact of encapsidated Staufen-2 alone or with its partners Staufen-1 or A3G on viral infectivity: HIV-1\(\Delta\)_vif_ viral particles were produced from Staufen-2 FLAG or A3G HA or Staufen-1 FLAG, or vector alone transfected HEK293T cells. 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 prepared from vector control cells. The presented values represent mean \(\pm\) standard deviations (error bars) for three independent experiments. Asterisks (***) indicates \(p\)-value \(\leq\) 0.0001. While the packaging of restriction factors such as APOBEC3 proteins into virions is a prerequisite for targeting the early post-entry phase of infection, it is interesting to note the packaging of dependency factors such as Staufen proteins, which work in favor of HIV-1 production in both producer and target cells. The RNA- as well as Gag-interactome, is conceivably complex. Therefore, it is possible to have several nonspecific RNA-protein and protein-protein molecular interactions during HIV-1 assembly, ultimately carrying them into viral particles during egression. Therefore, we created Staufen-2 knockout cells to understand their influence on viral infectivity and production. The results conclusively demonstrate that the absence of Staufen-2 (to a lesser extent, Staufen-1 too) impairs the virion production and their infectivity. This highlights a new role of virion-bound Staufen proteins in promoting HIV-1 infection, likely during early post-entry events. It was also interesting to observe that Staufen-2, when packaged in an HIV-1\(\Delta\)_vif_, caused increased infectivity (Figure 6), a finding that may justify further investigation. We observed the packaging of Staufen-2 into SIVagm virions (Supplementary Figure S5), which suggests that encapsidation of RNA-binding proteins may be a more fundamental phenomenon. This raises the likelihood of Staufen-2's interaction with other retroviruses such as murine leukemia virus (MLV) and other RNA viruses, although this remains to be elucidated. Our results can only postulate that the mechanism(s) that transports cellular Staufen-2 to HIV-1 and SIVagm are likely to be similar. According to the BioGRID database (thebiogrid.org/117978, which maintains curated entries of biological interaction based on original publications), there are 108 protein interactors of Staufen-2, including Staufen-1, MOV10 RNA helicase, HIV-1 Tat, ADAR RNA adenosine deaminase, and nucleocapsid of SARS-CoV-2. In addition, a previous study using mass spectrometry identified 45 proteins (22%) from Staufen-1 associated RNPs, which were found in HIV-1 particles, suggesting that RNP complexes could likely serve as vehicles [61].