# Encapsidation of Staufen-2 Enhances Infectivity of HIV-1
## 4 Discussion
These data prompted us to study Staufen-2 interaction with the host and HIV-1 proteins, and we included HIV-1 Gag, Staufen-1, and A3G to explore the underlying modes of viral packaging. As anticipated, Staufen-2 interacted with Gag and is probably responsible for its packaging into HIV-1. Additionally, its interaction with other host proteins such as Staufen-1 and A3G, characterized as RNP complex components, is consistent. This evidence reports an independent binding of Staufen-2 with Staufen-1 and A3G, which hypothetically raises the possibility of different modes of viral incorporation and, consequently, their synergetic action in HIV-1 replication. Certainly, follow-up studies using single virion microscopy to determine co-packaging rates and detailed mutational analysis to identify responsible residues mediating interactions may aid in their validation. Our results propose a model in which Staufen-2 packaging into HIV-1 is mediated by its interaction with Gag and probably independent binding of factors such as a positive effector, Staufen-1, or a negative (restriction) effector, A3G, collectively accelerates its viral encapsidation. Since the distribution of host factors during packaging is dynamic, different virions emerging from an infected cell are likely to carry varying compositions of host protein contributing to a pool of proteomically divergent virions (quasispecies) [68; 69; 70]. This differential host protein packaging may explain the basis of differential infectivity of emerging virions from different cell types, cellular microenvironment, factors that stimulate restriction factors (cytokine environment of the infected cell), depleting proviral factors, which together would decide the fate of viral infectivity and disease progression. Hence, the viral populations studied here would be expected to contain differential amounts of packaged proteins in diverse combinations such as Staufen-2 only, or Staufen-2 associated with Staufen-1/A3G, or either of one factor or even none. This perspective also thrusts an evolutionary advantage to the viral population carrying predominantly positive effectors such as Staufen and disadvantage to the viral population carrying predominantly negative effectors such as A3G. Overall, these factors can drive the viral population dynamics, infectivity, and evolution within a patient deciding the fate of disease progression. Moreover, there is a likelihood of involvement of Staufen-2 in Staufen-1-mediated processes during HIV infection, such as disruption of stress granule formation [12; 13]. The increase in the infectivity of the viral population with encapsidated Staufen-2 may be speculated due to increased protection provided to HIV-1 RNA against RNA recognition and degradation by host sensors. In addition, conceptually there is a possibility of Staufen-2's function in nuclear import, and RT. The additional roles of Staufen-2 may be clarified by identifying Staufen-2 residing viral compartment. Further, as phenylalanine glycine motif (FG-motif) containing proteins, such as Sec24C, tends to increase the HIV-1 capsid stability [71], FG-motif present in Staufen-2 RNA-Binding-Domain 2 (Uniprot Q9 NUL3) [19] may also function similarly, contributing to increased viral infectivity. It is interesting to note that while packaging of Staufen-2 enhances the viral infectivity, HIV-1 produced from cells expressing A3G/Staufen-2, the infectivity of the viral population is reduced (Figure 6). The result indicates that the antiviral activity of A3G could not be entirely overcome by either Staufen-2 alone or Staufen-2 interacting with A3G. Similarly, we also expected that Staufen-2 would not have any inhibitory effect on either the stability or enzymatic activity of A3G. Our study supports that Staufen-2 neither protects A3G from Vif-mediated degradation (Supplementary Figure S6A-C) nor inhibits the cytidine deaminase activity of A3G (Supplementary Figure S6D,E). These observations rationalize why the antiviral impact of A3G prevails, even when likely packaged with Staufen-2 (Figure 6).