Molecular Dynamics Simulation for Probing the Flexibility of the 35 Nucleotide SL1 Sequence Kissing Complex from HIV-1Lai Genomic RNA

Abstract

The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411–419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1₃₅ KC model which was submitted to a 31 ns molecular dynamics simulation (MD).

Our results allows to describe the internal dynamics of SL1₃₅ KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.     

Base pairing at the stem-loop junction in the SL1 kissing complex of HIV-1 RNA: a thermodynamic study probed by molecular dynamics simulation

The thermodynamics of the opening/closure process of a GC base pair located at the stem-loop junction of the SL1 sequence from HIV-1(Lai) genomic RNA was investigated in the context of a loop-loop homodimer (or kissing complex) using molecular dynamics simulation. The free energy, enthalpy and entropy changes for the closing reaction are 0 kcal x mol(-1), -11 kcal x mol(-1) and -0.037 kcal x mol(-1) x K(-1) at 300 degrees K respectively. Furthermore it is found that the free energy change is the same for the formation of a 11 nucleotide loop closed with UG and for the formation of a 9 nucleotide loop closed with GC. Our study evidences the high flexibility of the nucleotides at the stem-loop junction explaining the weak stability of this structure.     

A Molecular Dynamics Simulation Study of Coaxial Stacking in RNA

Abstract

We report on unrestrained molecular dynamics simulations of an RNA tetramer binding to a tetra-nucleotide overhang at the 5′-end of an RNA hairpin (nicked structure) and of the corresponding continuous hairpin with Na⁺ as counterions. The simulations lead to stable structures and in this way a structural model for the coaxially stacked RNA hairpin is generated. The stacking interface in the coaxially stacked nicked hairpin structure is characterized by a reduced twist and shift and a slightly increased propeller twist as compared to the continuous system. This leads to an increased overlap between C22 and G23 in the stacking interface of the nicked structure. In the simulations the continuous RNA hairpin has an almost straight helical axis. On the other hand, the corresponding axis for the nicked structure exhibits a marked kink of 39°. The stacking interface exhibits no increased flexibility as compared to the corresponding base pair step in the continuous structure.     

cis-Acting determinants of 7SL RNA packaging by HIV-1

The host noncoding RNA 7SL is highly enriched in the virions of retroviruses. We examined the regions of 7SL that mediate packaging by HIV-1. Both the Alu domain and the S domain were sufficient to mediate specific packaging when expressed separately as truncations of 7SL. However, while the Alu domain competed with endogenous 7SL for packaging in proportion to Gag, the S domain was packaged additively, implying that the Alu and S domains are packaged via separate mechanisms and that the Alu domain is packaged by the same mechanism as endogenous 7SL. Further truncations of the Alu domain or mutation of the Alu domain helix 5c region significantly reduced packaging efficiency, implicating helix 5c as critical for packaging, reinforcing the finding that 7SL packaging is highly selective, and confirming that 7SL is not passively acquired. Surprisingly, when the Alu domain was mutated so that it no longer contained a binding site for the SRP protein heterodimer SRP9/14, it was no longer packaged in a competitive manner but instead was packaged additively with endogenous 7SL. These data support a model in which 7SL RNA is packaged via interactions between Gag and a 7SL RNA structure that exists transiently at a discrete stage of SRP biogenesis. Our data further indicate that a secondary "additive" pathway exists that can result in the packaging of certain 7SL derivatives in molar excess to endogenously packaged 7SL.     

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg²⁺ ion at site 1 (Mg₁ ²⁺). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg₁ ²⁺ ion, coupled with the Mg₂ ²⁺ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg₁ ²⁺ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg₁ ²⁺ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg₁ ²⁺ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.     

Modeling the dynamics of the solvated SL1 domain of HIV-1 genomic RNA

A mode-coupling solution of the Smoluchowski diffusion equation (MCD theory), designed to describe the dynamics of wobbling macromolecules in water, is applied to a macromolecular bead model including water beads in the nearest layers. The necessary statistical averages are evaluated by time averaging along a molecular dynamics (MD) trajectory where both solute and water are introduced as atomistic models. The cross peaks in (1)H nuclear Overhauser effect spectroscopy (NOESY) NMR spectra that are routinely measured to determine biological structures are here calculated for the mutated 23 nucleotides stem-loop fragment of the SL1 domain in the HIV-1(Lai) genomic RNA. The calculations are in acceptable agreement with experiments without requiring any screening of the hydrodynamic interactions. The screening of hydrodynamics was necessary in previous MCD calculations obtained by using the same full atomistic MD trajectory, but a nonsolvated frictional model.     

SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA

Background

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of HIV is located on the 5′ end of the viral genome and suggested to form complex secondary/tertiary structures. Within this structure, stem-loop 1 (SL1) is believed to be most important and an essential key to dimerization, since the sequence and predicted secondary structure of SL1 are highly stable and conserved among various virus subtypes. In particular, a six-base palindromic sequence is always present at the hairpin loop of SL1 and the formation of kissing-loop structure at this position between the two strands of genomic RNA is suggested to trigger dimerization. Although the higher-order structure model of SL1 is well accepted and perhaps even undoubted lately, there could be stillroom for consideration to depict the functional SL1 structure while in vivo (in virion or cell).

Results

In this study, we performed several analyses to identify the nucleotides and/or basepairing within SL1 which are necessary for HIV-1 genome dimerization, encapsidation, recombination and infectivity. We unexpectedly found that some nucleotides that are believed to contribute the formation of the stem do not impact dimerization or infectivity. On the other hand, we found that one G–C basepair involved in stem formation may serve as an alternative dimer interactive site. We also report on our further investigation of the roles of the palindromic sequences on viral replication. Collectively, we aim to assemble a more-comprehensive functional map of SL1 on the HIV-1 viral life cycle.

Conclusion

We discovered several possibilities for a novel structure of SL1 in HIV-1 DLS. The newly proposed structure model suggested that the hairpin loop of SL1 appeared larger, and genome dimerization process might consist of more complicated mechanism than previously understood. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.

     

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.     

Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage

Initiation of retroviral genomic RNA dimerisation is mediated by the mutual interaction of the dimerisation initiation site (DIS) stem–loops near to the 5′ end of the RNA. This process is thought to involve formation of a transient ‘kissing’ complex over the self-complementary loop bases, which then refolds into a more stable extended interaction. We have developed a novel experimental system that allows us to clearly detect the extended duplex in vitro. Ribozyme sequences were incorporated into or adjacent to the type 1 human immunodeficiency virus DIS stem, leading to the formation of a functional ribozyme only in the extended duplex conformer. Here we show that extended duplex formation results in ribozyme cleavage, thus demonstrating the double-stranded nature of the extended complex and confirming that refolding occurs via melting of the DIS stems. Loop complementarity is essential for extended duplex formation but no sequence requirements for the loops were observed. Efficiency of extended duplex formation is dependent on the strength of the loop–loop interaction, temperature, the magnesium concentration and is strongly accelerated by the viral nucleocapsid protein NCp7. Our ribozyme-coupled approach should be applicable to the analyses of other refolding processes involving RNA loop–loop interactions.     

Structure of SL4 RNA from the HIV-1 packaging signal.

The NMR-based structure is described for an RNA model of stem-loop 4 (SL4) from the HIV-1 major packaging domain. The GAGA tetraloop adopts a conformation similar to the classic GNRA form, although there are differences in the details. The type II tandem G.U pairs have a combination of wobble and bifurcated hydrogen bonds where the uracil 2-carbonyl oxygen is hydrogen-bonded to both G,H1 and G,H2. There is the likelihood of a Na(+) ion coordinated to the four carbonyl oxygens in the major groove for these G.U pairs and perhaps to the N7 lone pairs of the G bases as well. A continuous stack of five bases extends over nearly the whole length of the stem to the base of the loop in the RNA 16mer: C15/U14/G13/G5/C6. There is no evidence for a terminal G.A pair; instead, G1 appears quite unrestrained, and A16 stacks on both C15 and G2. Residues G2 through G5 exhibit broadened resonances, especially G3 and U4, suggesting enhanced mobility for the 5'-side of the stem. The structure shows G2/G3/U4 stacking along the same strand, nearly isolated from interaction with the other bases. This is probably an important factor in the signal broadening and apparent mobility of these residues and the low stability of the 16mer hairpin against thermal denaturation.     

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.     

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ~2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca²⁺ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.     

Molecular dynamics simulation for probing the flexibility of the 35 nucleotide SL1 sequence kissing complex from HIV-1Lai genomic RNA

The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411-419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1(35) KC model which was submitted to a 31 ns molecular dynamics simulation (MD). Our results allows to describe the internal dynamics of SL1(35) KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.     

Insight into Signal Transduction: Structural Alterations in Transmembrane Helices Probed by Multi-1 ns Molecular Dynamics Simulations

Abstract

The hypothesis of structural alteration in transmembrane helices for signal transduction process is viewed by molecular dynamics simulation techniques. For the c-erbB-2 transmembrane domain involved in oncogenicity, the occurrence of conformational changes has been previously described as transition from the α to π helix. This dynamical feature is thoroughly analyzed for the wild phenotype and oncogenic sequences from a series of 18 simulations carried out on one nanosecond time scale. We show that these structural events do not depend upon the conditions of simulations like force field or starting helix coordinates. We demonstrate that the oncogenic mutations Val659 Glu, Gin and Asp do not prevent the transition. Furthermore, we show that β branched residues, in conjunction with Gly residues in the c-erbB-2 sequence, act as destabilizers for the α helix structure, π deformations are tightly related to other local structural motifs found in soluble and membrane proteins. These structural alterations are discussed in term of structure-activity relationships for the c-erbB-2 activating mechanism mediated by transmembrane domain dimerization.     

Modeling the dynamics of a mutated stem-loop in the SL1 domain of HIV-1Lai genomic RNA by 1H-NOESY spectra

The cross-peaks of ¹H-NOESY spectra at different time delays are compared to a mode-coupling diffusion (MCD) calculation, including the evaluation of the full ¹H relaxation matrix, in the case of a 23 nucleotide fragment of the stem-loop SL1 domain of HIV-1_Lai genomic RNA mutated in a single position. The MCD theory gives significant agreement with ¹H relaxation experiments enabling a thorough understanding of the differential local dynamics along the sequence and particularly of the dynamics of nucleotides in the stem and in the loop. The differential dynamics of this hairpin structure is important in directing the dimerization of the retroviral genome, a fundamental step in the infectious process. The demonstration of a reliable use of time dependent NOE cross-peaks, largely available from NMR solution structure determination, coupled to MCD analysis, to probe the local dynamics of biological macromolecules, is a result of general interest of this paper.     

Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.     

Calculation of Energy for RNA/RNA and DNA/RNA Duplex Formation by Molecular Dynamics Simulation

Molecular Biology - The development of approaches for predictive calculation of hybridization properties of various nucleic acid (NA) derivatives is the basis for the rational design of the...     

Molecular Insight into the Conformational Dynamics of the Elongin BC Complex and Its Interaction with HIV-1 Vif

The human immunodeficiency virus type 1 virion infectivity factor (Vif) inhibits the innate viral immunity afforded by the APOBEC3 family of cytidine deaminases. Vif targets the APOBEC3 family for poly-ubiquitination and subsequent proteasomal degradation by linking the Elongin-BC-dependent ubiquitin ligase complex with the APOBEC3 proteins. The interaction between Vif and the heterodimeric Elongin BC complex, which is mediated by Vif's viral suppressor of cytokine signaling box, is essential for Vif function. The biophysical consequences of the full-length Vif:Elongin BC interaction have not been extensively reported. In this study, hydrogen exchange mass spectrometry was used to dissect the Vif:Elongin BC interaction. Elongin C was found to be highly dynamic in the Elongin BC complex while Elongin B was much more stable. Recombinant full-length Vif interacted with the Elongin BC complex in vitro with a K_d of 1.9 μM and resulted in observable changes in deuterium uptake in both Elongin C and B. Upon binding to Elongin BC, no significant global conformational changes were detected in Vif by hydrogen exchange mass spectrometry, but a short fragment of Vif that consisted of the viral suppressor of cytokine signaling box showed decreased deuterium incorporation upon Elongin BC incubation, suggesting that this region folds upon binding.