**9. Cellular Factors Interacting with the Rev and the RRE**

While this review has concentrated on the dynamic nature of the HIV-1 and HIV-2 RREs, it is important to emphasize that concerted binding of various cellular factors is required for Rev-RRE function, most of which promote shuttling of Rev and RRE across the nuclear membrane. Two key factors are Crm1 [25,78–80] and importin β [81,82]. Crm1, or Exportin-1, is a member of the β-importin family of transport receptors and has been demonstrated to specifically interact with many proteins containing NES domains. Normally employed for the nucleocytoplasmic transport of proteins, snRNAs and rRNAs, the Crm1 pathway is hijacked by HIV to facilitate export of intron-retaining RNAs. Crm1 serves as an adaptor for binding of Ran-GTP [83–85] and nucleoporins such as Rip/Rab [86] to Rev, forming a functional export complex that ferries the Rev/RRE RNP across the nucleopore complex into the cytoplasm. In the cytoplasm, RanGAP1 and RanBP1 hydrolyze Ran-GTP to Ran-GDP, releasing

Crm1 from the Rev/RRE complex [86]. Importin-β then binds to the Rev NLS region, facilitating displacement of the RRE for translation of viral transcripts. The resulting Rev/importin-β complex interacts with Ran-GDP, allowing translocation of the Rev complex into the nucleus. Inside the nucleus, RCC1 catalyzes conversion of Ran-GDP to Ran-GTP, leading to dissociation of Rev from importin-β [87]. This dissociation unmasks the Rev NLS domain, promoting interaction of its NLS/RBD region with the RRE. Several members of the DEAD box RNA helicase family, such as RHA [88], DDX3 [89], DDX1 [90,91], DDX5 [92] and DDX24 [93], have also been reported as cellular co-factors of Rev, although their precise role in the Rev/RRE pathway remains to be elucidated. Most of these are nucleocytoplasmic shuttling proteins some of which have been implicated in promoting HIV genome packaging, restriction of Rev function in astrocytes, premature release of intron-retaining HIV transcripts from the splicing machinery. Similarly, cellular factors such as eIF-5A, SF2/ASF, B23, p32, Sam68, and ATM kinase have been proposed to modulate Rev/RRE function, though their roles are either unclear or controversial [38,94].

### **10. Conclusions and Outlook**

Although the role of the RRE in directing nucleocytoplasmic transport of unspliced and partially spliced viral RNAs has been unchallenged for over three decades, the structural flexibility that mediates these steps in HIV replication is only now coming to light, in many cases, following the development of new RNA probing strategies. For example, the generally accepted notion that RRE SL-I played a structurally "passive" role has now been challenged by chemical and biophysical techniques suggesting it folds into the central RRE core, possibly as a check for Rev loading. By extending early data on the structure of RRE-61, we now have evidence that the wild-type RRE may, in fact, exist as an equilibrium of conformers, each of which confers distinct growth properties in cell culture that may extend as far as their evolution in HIV-infected individuals. One might speculate that RRE structural fluidity would confer growth/fitness advantage to the virus by acting as "replication rheostat", regulating the level of HIV gene expression by allowing RRE to adopt various functional states. A less active RRE would shield HIV from a functional host immune system during early stages of infection, allowing the virus to establish itself within the host. Conversely, highly active RREs would promote a higher rate of virus replication without CTL killing in immunocompromised individuals. Unlike mechanisms that affect overall transcription and translation, down-modulation of gene expression by less active RRE conformers primarily affects viral structural protein expression and is not expected to affect HIV Nef expression. Thus, this mechanism would allow the immune evasion activities of Nef to persist, while the lower structural protein levels would make the infected cells less susceptible to CTL killing, As we uncover additional aspects of RRE conformational dynamics, exploiting this vis-à-vis developing novel high throughput screening strategies, exemplified here with branched peptides and small molecules that recognize the viral epitranscriptome, promises to open new and exciting avenues for targeting viral RNA genomes.

**Acknowledgments:** SFJLG and CS were supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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