Altered‐specificity mutants of the HIV Rev arginine‐rich motif‐RRE IIB interaction

Arginine‐rich motifs (ARMs) bind RNA structures with high affinity and specificity, and the human immunodeficiency virus (HIV) exploits ARM‐RNA interactions to regulate its lifecycle. The expression of HIV structural genes relies on recognition between the ARM of its Rev protein and its primary binding site, an internal loop in the viral RNA, the Rev‐response element region IIB (IIB). Many functional variants of the Rev ARM‐IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. A double mutant of Rev ARM, R35G‐N40 V, uses an unknown strategy to recognize IIB. Here, isothermal titration calorimetry and gel shift assays show that the R35G‐N40V‐IIB interaction has high affinity and specificity in vitro and a larger unfavorable entropy change upon binding than that of wild‐type Rev ARM‐IIB. In stark contrast with the critical dependence of wild‐type Rev on Arg35, Arg39, Asn40, and Arg44, mutational profiling shows R35G‐N40V is highly mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Affinity measurements in vitro and reporter assay measurements in vivo are consistent with the wild‐type Rev ARM and R35G‐N40V maintaining their recognition strategies when binding IIB mutants specific to wild‐type Rev ARM and R35G‐N40V, respectively. Some single amino acid mutants of wild‐type Rev ARM and R35G‐N40V have enhanced specificity, recognizing mutant IIBs yet not wild‐type IIB. These results provide another example of viral ARM‐RNA interactions evolving new specificities with few mutations, consistent with neutral theories of evolution.     

Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.     

Assignment and modeling of the Rev Response Element RNA bound to a Rev peptide using 13C-heteronuclear NMR

Summary The Rev Response Element (RRE) RNA-Rev protein interaction is important for regulation of gene expression in the human immunodeficiency virus. A model system for this interaction, which includes stem IIB of the RRE RNA and an arginine-rich peptide from the RNA-binding domain of Rev, was studied using multidimensional heteronuclear NMR. Assignment of the RNA when bound to the peptide was obtained from NMR experiments utilizing uniformly and specifically ¹³C-labeled RNA. Isotopic filtering experiments on the specifically labeled RNA enabled unambiguous assignment of unusual nonsequential NOE patterns present in the internal loop of the RRE. A three-dimensional model of the RNA in the complex was obtained using restrained molecular dynamics calculations. The internal loop contains two purine-purine base pairs, which are stacked to form one continuous helix flanked by two A-form regions. The formation of a G-G base pair in the internal loop requires an unusual structure of the phosphate backbone. This structural feature is consistent with mutational data as being important for the binding of Rev to the RRE. The G-G base pair may play an important role in opening the normally narrow major groove of A-form RNA to permit binding of the Rev basic domain.     

Single-nucleotide changes in the HIV Rev-response element mediate resistance to compounds that inhibit Rev function

Previously we described the identification of two compounds (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide [103833] and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile [104366]) that interfered with HIV replication through the inhibition of Rev function. We now describe resistant viral variants that arose after drug selection, using virus derived from two different HIV proviral clones, NL4-3 and R7/3. With HIV(NL4-3), each compound selected a different single point mutation in the Rev response element (RRE) at the bottom of stem-loop IIC. Either mutation led to the lengthening of the stem-loop IIC stem by an additional base pair, creating an RRE that was more responsive to lower concentrations of Rev than the wild type. Surprisingly, wild-type HIV(R7/3) was also found to be inhibited when tested with these compounds, in spite of the fact this virus already has an RNA stem-loop IIC similar to the one in the resistant NL4-3 variant. When drug resistance was selected in HIV(R7/3), a virus arose with two nucleotide changes that mapped to the envelope region outside the RRE. One of these nucleotide changes was synonymous with respect to env, and one was not. The combination of both nucleotide changes appeared to be necessary for the resistance phenotype as the individual point mutations by themselves did not convey resistance. Thus, although drug-resistant variants can be generated with both viral strains, the underlying mechanism is clearly different. These results highlight that minor nucleotide changes in HIV RNA, outside the primary Rev binding site, can significantly alter the efficiency of the Rev/RRE pathway.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

Specific binding of a basic peptide from HIV-1 Rev.

Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents.     

Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA

In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.     

Random mutagenesis and selection of organic solvent‐stable haloperoxidase from Streptomyces aureofaciens

Haloperoxidases are useful oxygenases involved in halogenation of a range of water‐insoluble organic compounds and can be used without additional high‐cost cofactors. In particular, organic solvent‐stable haloperoxidases are desirable for enzymatic halogenations in the presence of organic solvents. In this study, we adopted a directed evolution approach by error‐prone polymerase chain reaction to improve the organic solvent‐stability of the homodimeric BPO‐A1 haloperoxidase from Streptomyces aureofaciens. Among 1,000 mutant BPO‐A1 haloperoxidases, an organic solvent‐stable mutant OST48 with P123L and P241A mutations and a high active mutant OST959 with H53Y and G162R mutations were selected. The residual activity of mutant OST48 after incubation in 40% (v/v) 1‐propanol for 1 h was 1.8‐fold higher than that of wild‐type BPO‐A1. In addition, the OST48 mutant showed higher stability in methanol, ethanol, dimethyl sulfoxide, and N,N‐dimethylformamide than wild‐type BPO‐A1 haloperoxidase. Moreover, after incubation at 80°C for 1 h, the residual activity of mutant OST959 was 4.6‐fold higher than that of wild‐type BPO‐A1. Based on the evaluation of single amino acid‐substituted mutant models, stabilization of the hydrophobic core derived from P123L mutation and increased numbers of hydrogen bonds derived from G162R mutation led to higher organic solvent‐stability and thermostability, respectively. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:917–924, 2015     

The structure of the HIV-1 RRE high affinity rev binding site at 1.6 A resolution

The crystal structure of a 28 nt RNA fragment containing the human immunodeficiency virus type 1 (HIV-1) Rev response element high affinity binding site for Rev protein has been solved at 1.6 A resolution. The overall structure of the RRE helix is greatly distorted from A-form geometry by the presence of two purine-purine base-pairs and two single nucleotide bulges. G48 and G71 form a Hoogsteen-type asymmetric base-pair with G71 adopting a syn conformation. The non-canonical regions in the unliganded Rev response element molecule narrow the major groove width with respect to standard A-RNA. The Rev response element structure observed here represents a closed form of the Rev binding site and differs from conformations of the RNA observed previously by solution NMR studies.     

Continuous propagation of RRE(-) and Rev(-)RRE(-) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.

Molecular clones of human immunodeficiency virus type 1 that contained either 37 point mutations in the Rev-responsive element (RRE) that did not affect the overlapping env reading frame or both a mutated RRE and two mutations that eliminated Rev were constructed. The mutations in the RRE were shown to remove both negative and Rev-inducible positive effects of the RRE on gene expression (G. Nasioulas, A. S. Zolotukhin, C. Tabernero, L. Solomin, C. P. Cunningham, G. N. Pavlakis, and B. K. Felber, J. Virol. 68:2986-2993, 1994). Upon insertion of a cis-acting element of simian retrovirus type 1 (SRV-1) into these clones, both RRE(-) and Rev(-)RRE(-) clones were expressed efficiently. The element of SRV-1 has properties similar to those of the recently identified element of Mason-Pfizer monkey virus (M. Bray, S. Prasad, J. W. Dubay, E. Hunter, K.-T. Jeang, D. Rekosh, and M.-L. Hammarskjold, Proc. Natl. Acad. Sci. USA 4:1256-1260, 1994). We demonstrated that virus preparations produced after transfections of these SRV-1 element-containing molecular clones in human cells were infectious after cell-free transmission, that they replicated about 5 to 10 times less efficiently than wild-type virus, and that they were propagated continuously for more than 7 months in human peripheral blood mononuclear cells. Growth characteristics and sequence analysis of these viruses after long-term culture demonstrated that no RRE(+)Rev(+) revertants developed. These data demonstrate that human immunodeficiency virus type 1 Rev and RRE can be replaced by heterologous regulatory systems, resulting in efficient virus production. The resulting Rev(-)RRE(-) virus can be prepared and propagated efficiently in tissue culture and can be used for further studies of the life cycle of the virus. The data also suggest that Rev acts exclusively through the RRE interaction and that it does not have any additional essential function in the life cycle of the virus.     

Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.     

Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element

Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.