Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1

Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.     

Phosphorescence and optically detected magnetic resonance of HIV-1 nucleocapsid protein complexes with stem-loop sequences of the genomic Psi-recognition element

The binding of NCp7, the nucleocapsid protein of human immunodeficiency virus type 1, to oligonucleotide stem--loop (SL) sequences of the genomic Psi-recognition element has been studied using fluorescence, phosphorescence, and optically detected magnetic resonance (ODMR). RNA SL2, SL3, and SL4 constructs bind with higher affinity than the corresponding DNAs. G to I substitutions in the SL3 DNA loop sequence lead to reduced binding affinity and significant changes in the triplet state properties of Trp37 of NCp7, implicating these bases in contacts with aromatic amino acid residues of the zinc finger domains of NCp7, in agreement with the NMR structure of the 1:1 complex of NCp7 and SL3 RNA [DeGuzman, R. N., Wu, Z. R., Stalling, C. C., Pappaladro, L., Borer, P. N., and Summers, M. F. (1998) Science 279, 384-388]. The NCp7 to SL binding stoichiometry is 2:1 for intact SL sequences but is reduced to 1:1 for SL variants with an abasic or hydrocarbon loop. It is proposed that Delta D/Delta E(0,0), where Delta D is the change in the zero-field splitting D parameter and Delta E(0,0) is the shift of the tryptophan phosphorescence origin, provides a measure of aromatic stacking interactions with nucleic acid bases. Values on the order of 10(-5) indicate significant stacking interactions, while values closer to 10(-6) result from interactions not involving aromatic stacking. Binding of NCp7 to oligonucleotide substrates produces shortened Trp37 triplet state lifetimes by enhancement of k(x) and an increase of the relative value of P(x), the intersystem crossing rate to the T(x) sublevel. These effects are attributed to a reduction in the degree of electronic symmetry of Trp37 in the complexes. Guanine and adenine triplet states produced by optical pumping of SL3 DNA are characterized. We find, as with tryptophan, that D < 3E.     

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.     

Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA–protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.     

Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA

The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem-loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the microPsi RNA packaging signal. Results show that conservation of a long stem-loop structure and a loop-loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem-loop is sufficient to form an extended duplex and the whole stem-loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.     

Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 x 3 internal loop

The 5'-leader of HIV-1 RNA controls many viral functions. Nucleocapsid (NC) domains of gag-precursor proteins select genomic RNA for packaging by binding several sites in the leader. One is likely to be a stem defect in SL1 that can adopt either a 1 x 3 internal loop, SL1i (including G247, A271, G272, G273) or a 1 x 1 internal loop (G247 x G273) near a two-base bulge (A269-G270). It is likely that these two conformations are both present and exchange readily. A 23mer RNA construct described here models SL1i and cannot slip into the alternate form. It forms a 1:1 complex with NCp7, which interacts most strongly at G247 and G272 (K(d) = 140 nM). This demonstrates that a linear G-X-G sequence is unnecessary for high-affinity binding. The NMR-based structure shows an easily broken G247:A271 base pair. G247 stacks on both of its immediate neighbors and A271 on its 5'-neighbor; G272 and G273 are partially ordered. A bend in the helix axis between the SL1 stems on either side of the internal loop is probable. An important step in maturation of the virus is the transition from an apical loop-loop interaction to a dimer involving intermolecular interactions along the full length of SL1. A bend in the stem may be important in relieving strain and ensuring that the strands do not become entangled during the transition. A stem defect with special affinity for NCp7 may accelerate the rate of the dimer transformation. This complex could become an important target for anti-HIV drug development, where a drug could exert its action near a high-energy intermediate on the pathway for maturation of the dimer.     

HIV-1 nucleocapsid protein NCp7 and its RNA stem loop 3 partner: rotational dynamics of spin-labeled RNA stem loop 3

The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio or=3:1 complex also required intact Zn fingers. Stopped-flow EPR kinetics with NCP7/RNA mixed at a 4:1 ratio showed the major phase of NCp7 interaction with RNA stem loop 3 occurred within 4 ms, a second phase occurred with a time constant of approximately 30 ms, and a slower immobilization, possibly concomitant with large complex formation, proceeded over seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for viral packaging and genome fabrication.     

Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition.

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a approximately 110 nucleotide segment of the genome known as the Psi-site. The HIV-1 Psi-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity (K(d) approximately 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC (K(d)=(+/-14 microM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R=purine, N=G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Psi-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Psi-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.     

Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.     

197 Modeling the maturation pathway of the HIV-1 5’-UTR RNA dimer

HIV-1 genomic RNA is packaged as a dimer into the virions. The initial metastable RNA dimer is believed to be formed by virtue of “kissing interactions” between two copies of the palindromic apical loops of stem-loop SL1 of the 5’-untranslated region (5’-UTR) of the genomic RNA. Viral nucleocapsid protein NCp7 promotes maturation of the RNA dimer into more stable form, which involves extended or linear form of SL1 dimer (reviewed in Paillart et al., 2004; Moore & Hu, 2009; Lu et al., 2011). In vitro experiments have shown that this conversion occurs at stoichiometric amounts of NCp7 without breaking interactions between the two copies of the SL1 apical loops (Mujeeb et al., 2007). We have proposed a hypothetical pathway and calculated models of the intermediate structures for the SL1 stem-loop dimer maturation that does not require simultaneous dissociation of all base pairs in SL1 stems; this pathway involves formation of an RNA analog of the Holliday junction intermediate between the two stems of the SL1 dimer and a following branch migration towards the palindromic duplex (Ulyanov et al., 2011). Here, we extend these models to the dimer of the 1–344 fragment of HIV-1 RNA, which includes all of the 5’-UTR and the gag start AUG codon region, and show that the branch-migration mechanism of the dimer maturation is also feasible for the full 5’-UTR RNA. All RNA models have been calculated with the miniCarlo program (Zhurkin et al., 1991).     

hnRNP A1 binds promiscuously to oligoribonucleotides: utilization of random and homo-oligonucleotides to discriminate sequence from base-specific binding.

To understand the range of possible and probable A1 functions in pre-mRNA biogenesis, it is important that we quantify the relative ability (or inability) of A1 to bind high affinity RNA target sequences and/or structures. Using a fluorescence competition assay we have determined apparent binding affinities for a wide range of 20mer oligos containing putative and possible A1 targets including the high affinity 'winner' sequence identified by selection/amplification [Burd,C.G and Dreyfuss,G. (1994) EMBO J. 13, 1197-1204], AUUUA sequences found in 3'-UTRs of labile mRNAs, 5'- and 3'-splice sites and telomeric sequences. With the exception of a 20mer 'winner' sequence, all other 20mers examined bind A1 with a narrow, approximately 10-fold range of affinities extending from 3.2 x 10(6) to 4.2 x 10(7) M(-1). Studies with homo-oligomers suggest this range reflects nucleotide base rather than sequence specificity and hence, it was possible to predict reasonably accurate affinities for all other 20mers examined except for the 'winner', whose unusually high affinity of 4.0 x 10(8) M(-1) results from a unique higher order structure and sequence. Since there is no known physiological role for the 'winner' 20mer sequence, these data suggest A1 generally binds indiscriminately to all available pre-mRNA sequences. Both the large abundance of A1 in vivo and its binding properties are thus consistent with it playing a structural role in pre-mRNA biogenesis.     

Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7 g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7 g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7 g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7 g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.     

Mechanism of nucleocapsid protein catalyzed structural isomerization of the dimerization initiation site of HIV-1

Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.     

NMR structure of the complex between the zinc finger protein NCp10 of Moloney murine leukemia virus and the single-stranded pentanucleotide d(ACGCC): comparison with HIV-NCp7 complexes.

The structure of the 56 amino acid nucleocapsid protein NCp10 of retrovirus MoMuLV, which contains a single CX(2)CX(4)HX(4)C-type zinc finger, has been determined previously by NMR. The important role of NCp10 (or NCp7 for HIV-1) in the retroviral life cycle seems mainly related to their preferential binding to single-stranded nucleic acids. We report here the structure of the complex formed between the biologically active (14-53)NCp10 and the oligonucleotide d(ACGCC) in aqueous solution determined by 2D (1)H NMR based methods. The aromatic residue Trp(35) of NCp10 directs nucleic acid complexation as shown by its complete fluorescence quenching upon addition of d(ACGCC). (1)H and (31)P NMR studies support the insertion of Trp(35) between the G(3) and C(4) bases. A total of 577 NOE distance restraints, of which 40 were intermolecular, were used for the structure determination. The zinc finger provides a well-defined surface for the binding of d(ACGCC) through hydrophobic interactions and tryptophan stacking on the guanine. This latter interaction was also observed in the NMR-derived structures of the complexes between NCp7, which contains two successive zinc fingers, and single-stranded DNA and RNA, supporting the proposal for a major role played by aromatic residues of NCp proteins in nucleic acid recognition. Upon binding to the nucleotide a new loop in NCp10 that participates in the intermolecular interaction is formed. Additional interactions provided by positively charged residues surrounding the zinc finger appear necessary for tight binding. The structure of the complex NCp10-d(ACGCC) gives a structural explanation for the loss of virus infectivity following point mutations in the finger domain.     

RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome

The leader region of the human immunodeficiency virus type 1 (HIV-1) genome has a highly folded structure, comprising at least two RNA stem-loops [the transactivation response (TAR) and poly(A) hairpins] near its 5' end and four others (SL1 to SL4) downstream. Each of these stem-loops contributes to the function of the HIV-1 packaging signal, which efficiently targets genomic RNA into nascent virions. The central 140-base region of the leader, which includes the U5 and primer binding site (PBS) sequences, is also believed to adopt a complex structure, but the nature of this structure and its possible role in RNA packaging have not been extensively explored. Here we report a mutational analysis identifying at least three separate loci within the U5-PBS region which, when mutated, impair both HIV-1 packaging specificity and infectivity in a single-round proviral assay. In common with those of all previously described packaging signals in the leader, the function of one of these loci appeared to depend on secondary structure rather than on sequence alone. By contrast, the activity of the other two loci did not correlate with any predicted conformations. Moreover, unlike SL1 to SL4, the TAR, poly(A), and U5-PBS hairpins were not bound with high affinity by the nucleocapsid portion of the HIV-1 Gag protein in vitro, implying that they contribute to packaging through a mechanism distinct from that of SL1 to SL4. Our findings confirm the existence and importance of secondary structure around the PBS and demonstrate that functional packaging signals are distributed across the entire HIV-1 leader.