Acidic pKas of histidines buried within the protein interior are frequently rationalized on the contradictory basis of either polar interactions within the protein or the effects of a hydrophobic environment. To examine these relationships, we surveyed the buried surface area, depth of burial, polar interactions, and crystallographic temperature factors of histidines of known pKa. It has been found that buried environments of histidines do not always result in acidic pKas. Instead, the variability of histidine pKas increases for residues where the majority of the side-chain is buried. Because buried histidines are always found in mixed polar/apolar environments, multiple environmental contributions to pKa values must be considered. However, the quantitative relationships between heterogeneous environments and pKa values are not immediately apparent from the available data.

The HIV-1 Rev protein is essential for the virus because it promotes nuclear export of alternatively-processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in E. coli is a well-behaved folded protein. Binding assays using fluorescently-labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell based assays of HIV-1 production, and provide the first demonstration that recombinant DDX1 binds Rev and RNA, and has RNA dependent catalytic activity.

Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced HIV-1 viral mRNA transcripts. Several host factors, including the human DEAD-box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev-RRE complexes, in the presence or absence of DDX1, was observed in real-time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently-labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev:RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to non-productive nucleation events, resulting in increased occurrence of higher order Rev:RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (AMP-PNP), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and AMP-PNP, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE.

The translation of the unspliced and partially spliced viral mRNAs that encode the late, structural proteins of HIV-1 depends on the viral-protein Rev. Oligomeric binding of Rev to the Rev response element (RRE) in these mRNAs promotes their export from the nucleus and thus controls their expression. Here, we compared the effects of hydrophobic to hydrophilic mutations within the oligomerization domain of Rev using assays for oligomeric RNA binding, protein structure, and export from the nucleus. Oligomeric RNA binding alone does not correlate well with RNA transport activity in the subset of mutants. However, protein structure as judged by CD spectroscopy does correlate well with Rev function. The oligomeric assembly of Rev-L18T is impaired but exhibits minor defects in structure and retains a basal level of activity in vivo. The prevalence of L18T in infected individuals suggests a positive selection mechanism for L18T modulation of Rev activity that may delay the onset of AIDS.Keywords: HIV-1; Rev; RRE; nuclear export; viral latencyThe Rev protein of HIV-1 controls gene expression by promoting the nuclear export of unspliced and partially spliced viral mRNA (Malim et al. 1989b;Pollard and Malim 1998;Hope 1999). Rev recognizes these mRNAs through both the binding of an arginine-rich RNA binding domain (RBD) to a specific binding site within the Rev response element (RRE) (Malim et al. 1989a;Bogerd and Greene 1993) and the self-association of Rev monomers into higher-order oligomeric complexes on the RRE (Malim et al. 1989a;Bogerd and Greene 1993). It is this strong tendency to self-associate that has thwarted attempts to obtain structural information of full-length Rev at the atomic level. Understanding the essential contribution made by Rev oligomerization to influence the course of the viral infection (Malim and Cullen 1991;Madore et al. 1994;Mann et al. 1994) has been hampered by the resulting lack of detailed structural information on the oligomeric complexes. Many questions concerning the connection between Rev structure and oligomeric Rev-RRE assembly (Blanco et al. 2001;Havlin et al. 2007), the self-association tendency of Rev in the absence of its cognate RRE (Wingfield et al. 1991;Cole et al. 1993;Havlin et al. 2007), and the observation of Rev variants with

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