Exchange of the Basic Domain of Human Immunodeficiency Virus Type 1 Rev for a Polyarginine Stretch Expands the RNA Binding Specificity, and a Minimal Arginine Cluster Is Required for Optimal RRE RNA Binding Affinity, Nuclear Accumulation, and trans -Activation

Abstract

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.The Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1) is required for the expression of unspliced and partly spliced RNAs for the viral structural proteins (19,31,45). Rev modulates splicing, nuclear export, and cytoplasmic utilization of unspliced or partially spliced viral mRNAs (11,13,21,22,52,54,74) by binding to a highly structured Revresponsive element (RRE) RNA sequence embedded in the env mRNA (17,30,33,36,52,57,72,97). RRE RNA folds into four stem-loops designated A, C, D, and E or stem-loops I, III, IV, and V with a branched stem-loop structure (B, B1, or B2 [or stem-loop II A, B, or C]) linked by a central loop (36,52,57). Most of the RRE structure is dispensable for Rev activity, and a minimal structure composed of the B, B1, or B2 (or stem-loop II A, B, or C) subdomain was active both in vitro and in vivo (37,41). Rev is a basic phosphoprotein that shuttles between the nucleus and the cytoplasm (24,42,61,70,95) but accumulates in the nucleus, concentrating in the nucleolus under steady-state conditions. Like many v...

A Solution to Limited Genomic Capacity: Using Adaptable Binding Surfaces to Assemble the Functional HIV Rev Oligomer on RNA

“…The tight correlation between in vitro binding and RNA export data (Figure 6F) suggests that the primary role of Rev oligomerization may simply be to enhance RNA-binding affinity. Consistent with this view are the findings that a single Rev NES is sufficient to mediate nuclear export (Wen et al, 1995) and that unspliced mRNAs can be exported even without Rev oligomerization if a second RRE-binding domain is covalently appended to Rev (Furnes et al, 2005) or if protein-RNA affinity is enhanced using the MS2 coat protein-RNA interaction (Nam et al, 2001). This manner of forming an RNP may also be utilized by Rev for functions other than RNA export, such as translation and genome packaging.…”

The HIV-1 Rev response element

“…Studies with chimeric Rev and RRE (Rev fused to MS2 coat-protein and RRE stem IIB replaced with the MS2-RNA) 33 - 35 and with polyvalent Rev-binding elements (e.g., stem IIB concatemers) 36 , 37 have shown that although high affinity correlates with function, affinity alone is unable to recapitulate full export activity of the Rev-RRE complex.…”

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

“…In support of this hypothesis, we have shown for example that polyQ or polyA repeats can mediate interactions between proteins that contain them (e.g., Fiumara et al, 2010 ; Pelassa et al, 2014 ), while polyproline (polyP)-II structures and proline-rich sequences can mediate protein-protein interactions by binding to non-repetitive interaction domains (Yu et al, 1994 ). Evidence exists that some charged AARs may also drive protein-nucleic acid and protein-lipid interactions (Dean, 1983 ; Nam et al, 2001 ; DeRouchey et al, 2013 ).…”