Structural Differentiation of the HIV-1 Poly(A) Signals

Transcription

1 Open Access Article The authors, the publisher, and the right holders grant the right to use, reproduce, and disseminate the work in digital form to all users. Journal of Biomolecular Structure & Dynamics, ISSN Volume 23, Issue Number 4, (2006) Adenine Press (2006) Structural Differentiation of the HIV-1 Poly(A) Signals Abstract The Human Immunodeficiency Virus Type 1 (HIV-1) encodes the polyadenylation (poly(a)) signal (AAUAAA) within the highly conserved untranslated region (UTR) at both 5 and 3 terminals of the viral transcript. In polyadenylation, an RNA transcript is cleaved and then elongated with adenine nucleotides while repression of the 5 signal and utilization of the 3 signal occurs. Because experimental studies have yet to analyze the structures of both 5 and 3 signals from a global perspective, other structural conformations involving these signals may exist and could be pivotal to understanding key functional processes. To distinguish the differential regulation of the 5 and 3 poly(a) signals, we studied the structural tendencies of both the 5 and 3 UTR in HIV-1. Through computational folding predictions of multiple HIV-1 strains using the Massively Parallel Genetic Algorithm (MPGAfold) capable of dynamically elucidating key alternative conformations, the 5 poly(a) signal was found to be dominantly occluded in a hairpin loop while the 3 poly(a) signal showed variability between hairpin and linear conformations with a propensity for the linear structure with an asymmetric internal loop. Furthermore, the energies and predictions of these structures indicate that the poly(a) signals have some metastable characteristics indicating an ability to switch into different conformations that can regulate viral function. Alan H. Gee 1 Wojciech Kasprzak 2 Bruce A. Shapiro 1,* 1 Center for Cancer Research Nanobiology Program National Cancer Institute Building 469, Room 150 NCI-Frederick Frederick, MD 21702, USA 2 Basic Research Program SAIC Frederick, NCI-Frederick Building 469, Room 150 Frederick, MD 21702, USA Key words: HIV-1; Massively parallel genetic algorithm; Metastable structures; Polyadenylation signal; and RNA structure. Introduction Upon entering a host cell, the RNA genome of HIV-1 undergoes reverse transcription, which produces proviral DNA that is subsequently integrated into a cell genome. After integration in the double-stranded DNA, the genomic DNA is then transcribed to make viral RNA. All HIV-1 viral RNA sequences contain a repetitive region at the 5 and 3 ends called the untranslated region (UTR) (Figure 1). These UTRs contain key regulatory signals that are recognized by the cell s transcription factors. Within the 5 UTR, the upstream terminal repeat (R) element contains several controlling functional motifs including the TAR (trans-activator response), a hairpin stem-loop structure that interacts with the Tat protein to stimulate RNA transcription (1), and the polyadenylation (poly(a)) hexamer signal (AAUAAA), which can potentially interact with protein factors to initiate polyadenylation. This poly(a) signal is part of a highly conserved structure in various HIV and SIV isolates (2). The U5 region, located downstream of the 5 R region, contains two important signals: the primer activation signal (PAS) which works in conjunction with the primer binding site (PBS) in the regulation of reverse transcription (3, 4) and the GU-rich or U-rich sequence which is needed for polyadenylation. Further downstream of the 5 leader is the RNA dimer initiation signal (DIS) that is essential for the packaging of infectious viral particles (5, 6) and the major splice donor site (SD). The structural configurations of the DIS and especially the 5 poly(a) signal are important in the regulation of viral function. In past in vitro studies on the 5 end, the interaction concerning the 5 poly(a) and DIS signals showed two *Phone: (301) Fax: (301) bshapiro@ncifcrf.gov 417

2 418 Gee et al. structural conformations for the poly(a) signal: a linear long distance interaction (LDI) and a branched multiple hairpin structure (BMH) (7-9). Each of these structural conformations has an important impact on the function of the virus: the linear long distance interaction is detrimental to dimer formation whereas the branched structure is amenable to dimer formation. Identical to the 5 UTR, the 3 UTR, in addition, contains the upstream U3 region which is not present in the 5 UTR, the R region, and the downstream U5 region. Although the terminal 5 R and U5 regions are equivalent to the 3 R and U5 regions, the viral functions of the 5 and 3 ends are discrete: the 5 end promotes transcription while the 3 end terminates transcription through the association of cleavage and polyadenylation. Polyadenylation is a process that maturates the 3 end of mrna by attaching a series of adenine nucleotides downstream from the conserved hexamer sequence after recognition by various protein factors, specifically the cleavage and polyadenylation specificity factor (CPSF) which binds to the poly(a) signal (10, 11) and the cleavage stimulation factor (CStF) which binds to the GU-rich or U-rich sequence located in the U5 region (10-13). The addition of the adenine or poly(a) tail serves to stabilize the mrna thus preventing degradation and influencing translation. Polyadenylation also is functionally linked to and enhances the splicing of mrna (10, 14, 15). Several mechanisms have been proposed to address the positive and negative regulation of the 5 and 3 poly(a) signals. Studies have shown in vitro and in vivo that an upstream enhancer element (USE) is essential for efficient polyadenylation (16, 17). The USE, located in the U3 region, which is present only on the 3 terminal (Figure 1), has been proposed to initially act as a binding or entry site for CPSF (18). When transient opening of the hairpin occurs, CPSF binds to the exposed hexamer sequence. In contrast, a mechanism on the 5 terminal has been proposed to down-regulate polyadenylation. Here, because the viral RNA lacks the U3 region and structureinduced regulation may not be enough to fully inhibit polyadenylation, the binding of the major SD site to the U1 snrnp in the HIV-1 leader region has been proposed to repress 5 polyadenylation (18, 19). In addition, in vitro studies suggest that the partial occlusion of the poly(a) hexamer motif in a hairpin structure inhibits the binding of the CPSF (18) and represses 5 polyadenylation (18, 20, 21), but a sufficiently stable 5 hairpin structure in genomic RNA is essential for the packaging of the viral genomes into virions (22). Figure 1: Schematic representation that depicts the 5 and 3 ends of HIV-1 RNA. The 5 leader region is depicted with the major landmarks in the R and U5 region being illustrated. Although the 5 terminal regions are equivalent to the 3 end, the viral functions of the ends are discrete: the 5 end initiates transcription while the 3 end is associated with cleavage and polyadenylation of the transcript. Polyadenylation requires the availability of the poly(a) signal for CPSF binding and the presence of the downstream GU-rich or U-rich sequence for CStF to bind. Previous studies, however, have not analyzed the apparent dual role of the repetitious 5 and 3 sequences and the formation of both the 5 and 3 structures of the poly(a) signal from a global perspective. In this investigation, we studied the folding propensities of the 5 and 3 poly(a) signals by using the dynamic folding characteristics of the Massively Parallel Genetic Algorithm (MPGAfold) (23-25, 31-34). Sequences were constructed by splicing together the 5 end with the 3 end of HIV-1 after folding four nine kilobase viral genomes to determine selfcontained domains that enable folding in manageable compute times. In folds of several such HIV-1 construct sequences, the 5 poly(a) signal was found to have a propensity to be associated with a hairpin stem-loop structure whereas the

3 3 poly(a) signal was mostly associated with a linear asymmetric internal loop structure. Previous in vitro studies suggested that the weakening of the 5 poly(a) hairpin increases the efficiency of premature polyadenylation and stabilizing the hairpin loop structure decreases efficiency (18, 20, 21). Even though dominant motifs are exhibited, the 5 and 3 structures, as determined by thermodynamic calculations, are metastable and can exist as either hairpin or linear motifs. So, we propose that the 3 poly(a) signal is more effective for activating polyadenylation due to its propensity for the linear structure and its ability to transition between hairpin and linear states more readily than the 5 signal. 419 Structural Differentiation of the HIV-1 Poly(A) Signals Biological functional states do not always correspond to the minimum free energy structure, and the RNA molecule can pass through various intermediate states on its way to a final state, as was shown in (23, 25) and references therein. To capture these transformations, we have developed several RNA structure algorithms to help better predict, analyze, and visualize RNA secondary structures. In particular, the MPGAfold program is based on the biological theories of evolution and survival of the fittest. It is capable of predicting RNA secondary structure folding dynamics and producing significant RNA structural intermediates and alternate conformations of a given sequence by utilizing thermodynamic energy parameters as an objective function (24-27). Having the ability to incorporate some types of experimental data, it can both verify and predict the outcome of experiments by biasing or forcing known base pairs or varying the energy rules based on temperature and/or ionic conditions. STRUCTURELAB, an interactive RNA structure analysis workbench, has been helpful in data mining the large quantities of data produced by the folding program (9, 23, 25, 28, 29). Materials and Methods Construction of Sequences A total of nine HIV-1 sequences was obtained from the Los Alamos National Laboratory HIV sequence database: LAI, HXB2R, and MN from subtype B; CM240 and CF402 from subtype AE; IBNG from subtype AG; U455 from subtype A1; and NDK and ELI from subtype D. To evaluate the structure of the poly(a) signal on both ends of HIV-1, the RNA secondary structure foldings were analyzed by splicing together the 5 and 3 ends of the virus to obtain a more manageable sequence length. The domains for defining the connecting points of the 5 and 3 ends of our constructs (cut sites) were determined with Mfold (26), by folding the full RNA genomic sequence from the 5 R to 3 U5 region (approximately 9,000 nucleotides long) of four representative sequences from subtypes AE, AG, B, and D (CM240, IBNG, LAI, and ELI respectively). The 3 U5 region was included because polyadenylation requires the GU-rich or U-rich sequence present in the U5 region (12). A second form of the RNA (5 R to 3 R) sequence, which represents the genome after cleavage, was also folded using Mfold to ensure consistent domains before and after the cleavage of the transcript. The purpose of folding the full RNA sequences was to ensure that the cut sites determine well-defined domains that would not disrupt any structural motifs predicted in the entire genomic folds. The four sequences tested had optimal or near optimal structures that corresponded to the domains we found by folding both forms of the full genomic HIV-1. The USE motif, which was located prior to the cut sites and was consistent with our selfcontained domains, was not included in the constructs. Thus, the self-contained domains were consistent across different sequences of different subtypes. Based on the full genomic folds, the cut sites for the 5 and 3 ends were determined using alignments derived from the Los Alamos HIV Database. From the alignments, all the positions relative to LAI s position 662 and position 9129 were determined and cut (Table I). The 5 and 3 ends were then juxtaposed, creating manageable constructs. Two forms of the HIV-1 constructs were created to depict

4 420 Gee et al. the two stages during polyadenylation: one construct included the 5 R to 3 U5 region ( nucleotides) which represents the genome before cleavage and another construct included the 5 R to 3 R region ( nucleotides) which represents the genome after cleavage. Prediction of RNA Secondary Structures The secondary structure predictions of four full genomic sequences from each subtype (9,000+ nucleotides) were produced with Mfold, and the first one thousand suboptimal structures were examined with help of the STRUCTURELAB s Stem Trace to determine the conservation of the cut site motifs. In other respects, however, these folds were not very revealing, since the 5 and 3 poly(a) stems were generally present in a minority of solutions and were uniformly distributed throughout the top 1,000 structures, making it difficult to assess their relative propensity of occurrence. RNA secondary structure predictions of the constructs ( and nucleotide sequences) were performed using MPGAfold. Standard dynamic programming algorithms, such as Mfold or RNAfold (26, 30), in general have the ability to generate optimal and suboptimal conformers without indicating specific intermediate and lower fitness structures that precede the final conformations. They also force generation of new structures by the deterministic mechanism that is inherent in their traceback procedure. MPGAfold, on the other hand, focuses attention on statistically significant intermediate and final states, which can be produced by the usage of important structural motifs. Since many motifs can enter a maturing structure at the same time, several domains can fold simultaneously thus simulating multiple folding nucleation points. This process could also be captured by sequential folding (see below), which biases however the interactions that can occur. The ability to capture structural intermediates, the dynamic behavior of a folding molecule, and the propensity for RNA to fold into different states was illustrated in (24, 25). The details of MPGAfold are presented in (24, 31-34), but a general outline will be briefly presented here. The algorithm begins by generating an evolving population of RNA structures each containing population elements (PE). Generally, 4K, 8K, 16K, 32K, 64K, 128K, PEs are used in each calculation which are laid out in a two-dimensional toroidal mesh. Each PE is surrounded by eight neighbors creating a 3 3 window consisting of nine possible structures that the algorithm chooses from. Based upon a biased free energy criterion, two parents are selected from the 3 3 neighborhood, and from those two parents, two children are created by randomly mutating stems from the stem pool and recombining stems from the two parent structures. The child with the best fitness or lowest free energy becomes the center for the next 3 3 neighborhood. The algorithm iterates this process over several generations creating new ensembles of PEs at each generation. A convergence criterion, which

5 measures the stability of the population, terminates the iterations when stable conditions are reached. A higher population size in general will yield final results with less variation when capturing the RNA secondary structure that best represents the run (population consensus structure). At lower populations the algorithm normally will converge to solutions that are characteristic of functional intermediate states, and at higher populations the structures will pass through multiple intermediates on their way to final structural states. The free energies of each structure are determined by using an established energy rule set with efn2 coaxial stacking energy calculations (26, 27) applied at run time at every generation. 421 Structural Differentiation of the HIV-1 Poly(A) Signals MPGAfold was used to fold our constructed 5 R to 3 U5 and 5 R to 3 R sequences of our nine constructs. The algorithm was run with population sizes of 4K, 8K, 16K, 32K, 64K, and 128K, each twenty times, to capture final, intermediate and alternative structures. MPGAfold also has a sequential folding capability that simulates RNA folding during transcription or 3 processing. At each generation, a user-specified number of nucleotides is either removed or added to simulate the effect of RNA synthesis or transcription on folding. Sequential folding allows or eliminates stems if they do not fit the sequence size of the current generation. We investigated the effects of sequential folding on the 5 and 3 poly(a) structures of the described HIV-1 constructs. Sequential folding experiments were run with population sizes of 4K to 128K with the two forms of the HIV-1 LAI and CM240 constructs. These sequences were extended by one to four nucleotides per generation to determine the variability, if any, of generated structures as a function of the elongation speed. It should be noted that the elongation rates do not necessarily correspond to actual RNA synthesis rates. Analysis and Visualization of the RNA Secondary Structures STRUCTURELAB, an RNA/DNA structure analysis workbench developed by our laboratory, was used to help visualize and analyze the large amounts of data derived from the MPGAfold and the Mfold programs. STEM TRACE, a component of STRUCTURELAB, is a visualization interface that was used to depict the developing structures in a run or the final results of MPGAfold runs (25, 29). In essence, STEM TRACE is a two-dimensional plot of all unique helical stems from a solution set produced by any folding algorithm for a given sequence. The interactive twodimensional graph of a set of RNA structures, depicted in a STEM TRACE, can portray the intermediate or final structures from several different population runs in one presentation. Each position along the x-axis represents an individual structure. A vertical y-coordinate is assigned to each stem in a structure, which is represented by a triplet (5 3 stem-size). As new structures are added, each new unique stem is assigned the next integral position along the y-axis, while previously appearing stems in another structure are assigned a y position equal to their first appearance. As a result, horizontal color-coded bands are constructed by the addition of identical stems for multiple structures. The outputs from the two folding programs used were submitted to STEM TRACE for the analysis and drawing of the structures. Results Full RNA Genomic Foldings In order to understand the secondary structure conformations before and after the 3 cleavage event during polyadenylation, two forms of the full genomic HIV-1 sequence were folded with Mfold. One form included the 3 U5 region, normally cleaved during polyadenylation, and the other form did not include this region. Four HIV-1 sequences from different subtypes were folded to determine the validity of the cut sites for the juxtaposition of the 5 and 3 UTR. The foldings of the 5 R to the 3 U5 sequences consistently demonstrated two different conformations that utilized the 3 U5 nucleotides downstream of the 3 poly(a) signal. These bases were

6 422 Gee et al. mostly in a self-contained motif or paired to bases in the 5 gag region. Thus, the nucleotides downstream of the 3 R region rarely interfered with the other secondary structures of either the 5 or 3 UTR. We also verified that the cut sites for the self-contained domains were compatible with all the studied 5 and 3 poly(a) conformations in both versions of the four full genomic sequences. The cut sites used to create the constructs were preserved in the Mfold s optimal and nearly optimal structures of the full genomic sequences. The similarity of the MPGAfold predicted structural motifs found in the constructs, compared to the motifs predicted by Mfold in the corresponding regions of the full genomic folds, leads us to believe that our constructs represent a valid structural reduction of the problem. Structural Motifs of Poly(A) Signals Based upon the foldings of the two forms of HIV-1 constructs (one form with the 3 U5 region and the other form without the region), there are several distinct motifs for the 5 and 3 polyadenylation signal (AAUAAA). In vitro, the 5 poly(a) signal has been shown to exist in two forms corresponding to inactive and active states (7, 8, 20, 35). For polyadenylation to occur, the 3 poly(a) signal must be in an accessible conformation while the 5 poly(a) signal must be suppressed to avoid premature polyadenylation. Experimental results have demonstrated that the hairpin structure exists in vitro and is essential for the inaccessibility of the poly(a) signal (18, 20-22). Ex vivo studies have recently demonstrated that the 5 poly(a) hairpin exists dominantly in cells (36). In vitro studies have shown that the hairpin has the potential to inhibit translation by forming a long range pseudoknot with the matrix coding region (35). The poly(a) hairpin conformation occludes the initial two adenine bases in a stem while the remainder of the signal is exposed in a hairpin-loop (Figure 2A). Throughout foldings of several HIV-1 constructs, this hairpin structure was the most dominant motif for the 5 poly(a) signal (refer to Table II and Figure 4). Experimental studies have shown that the existence of the hairpin structure represses 5 polyadenylation while weakening the hairpin causes premature polyadenylation and short transcripts (21). The hairpin structure may Figure 2: (A) 5 LAI poly(a) signal in a hairpin loop with the initial two adenine bases paired in a stem. The rest of the sequence UAAA is exposed in a hairpin loop. This structure is more dominant and preferred in the final structures of a LAI 128K population run with a frequency of 70-80%. The structural presentation of the signal may inhibit the ability for CPSF to recognize the signal. This structure is also representative of the other HIV-1 sequences discussed in this study. (B) A depiction of the dominant linear structural motif that partially opens the 3 poly(a) signal in HIV-1 LAI. Of the AAUAAA hexamer signal, the initial two bases AA are exposed in an asymmetric internal loop, and the remainder of the signal is bound in weak, easily opened A-U base pairs. This 3 linear structure for the poly(a) signal appears 85% of the time in the final structures of the 128K population run. But, the 3 poly(a) signal shows a more transient ability to change between a hairpin structure to a dominant linear structure than does the 5 poly(a) signal. Point mutations, which decrease the propensity for the formation of this linear structure, are marked for NDK and IBNG. Again, this structure is representative of the other HIV-1 sequences discussed in this study.

7 sequester the signal by disrupting protein complex formations, specifically during the binding of the ternary RNA-CPSF-CStF complex to the poly(a) signal (18). The majority of the remaining poly(a) structures were in a less fit conformation, specifically the 5 linear and 3 hairpin poly(a) structure (marked in green in Figure 4), where the 5 poly(a) signal is exposed in an internal loop. Because the 5 poly(a) signal was dominantly in a hairpin motif across various population sizes, this conformation may have the stability required for the suppression of 5 polyadenylation, as mentioned in (21). This supports our notion that the 5 poly(a) signal is dominantly in a hairpin motif that inhibits premature polyadenylation. 423 Structural Differentiation of the HIV-1 Poly(A) Signals In contrast, our predictions indicate that the 3 poly(a) signal has a propensity to be associated with a linear structure where the initial two adenine bases of the signal are exposed in an asymmetric internal loop motif with the rest of the signal being associated with weak A-U base pairs (Figure 2B). Present in both the full genomic and the constructed 5 and 3 foldings, the 3 linear poly(a) motif occurs in structures with the best fit energies and appears to be more thermodynamically stable than the 3 poly(a) hairpin structure. Out of one hundred twenty final structures with population sizes of 4K to 128K for the 3 U5 constructs, the 3 linear poly(a) motif had a frequency of 60% for LAI and 78.3% for CM240. Similar results and trends occur with the other sequences tested (see Table II). For all the 3 R constructs, the frequency for the 3 linear poly(a) signal was greater than 80% over populations of 4K to 128K, except IBNG, which was greater than 70% (data not shown). Because in vitro studies have shown that destabilization of the 5 poly(a) hairpin is capable of activating polyadenylation (20, 21) and an alternative structure to the 5 poly(a) hairpin is an internal loop (7-9), we propose that the linear 3 poly(a) signal, which is associated with an internal loop and shows variability between hairpin and linear structures, may provide the necessary opening of the signal to trigger 3 polyadenylation. Also, in vitro studies have shown that a stable 3 poly(a) hairpin can efficiently inhibit polyadenylation (21). Thus, the dominant linear 3 poly(a) signal motif which is dominantly associated with an internal loop may allow for effective activation of polyadenylation. Metastability in the Conformations of Poly(A) Signals In addition to having a dominant structure, the poly(a) signal has the ability to exist in two conformations: a hairpin loop structure or a linear stem structure (7, 8).

8 424 Gee et al. The MPGAfold results for various sequences were concatenated into one STEM TRACE that displayed the six distinct populations (Figure 3A). For the 5 Poly(A) signal, the less dominant structure is the linear poly(a) motif. As the population size increases, the more energetically stable structure, the 5 poly(a) hairpin, becomes more dominant. There is still some evidence of the linear 5 poly(a) structure, A. HIV-1 LAI 4K 8K 16K 32K 64K 128K 3 3 Hairpin poly(a) 8K HIV-1 CM240 16K 32K 64K 128K 3 Linear poly(a) STEMS (5, 3, size) Figure 3: (A) STEM TRACE of two different HIV-1 sequences at six different MPGAFold population sizes. The y-axes represent stems in the structures, and the xaxes represent the final structures (20 per population). The color bands represent the cumulative frequency of stems across all population bins. The size of the sequences is roughly nucleotides but only stems involving interactions between the 5 and 3 ends are shown. Shifts between poly(a) motifs are marked. (B) Folding prediction for the consensus structure of the LAI sequence (846 nt). This structure s energy is within 2% of the structure with the best energy, which contains both linear 5 and 3 poly(a) structures. 4K 5 Hairpin poly(a) 5 STRUCTURES STRUCTURES spliced cut sites B TAR 5 Hairpin poly(a) 3 Linear poly(a) PAS HIV-1 LAI (846 nt) E = kcal/mol, (128K) HIV-1 LAI Poly(A) Signal Conformations Energy ( kcal/mol) Figure 4: Distribution of 5 and 3 poly(a) motifs for HIV-1 LAI with the 3 U5 region of all final structures for the populations 4K to 128K sorted by the fitness of the structure. Each color represents a different set of poly(a) signal motifs: blue represents structures with 5 and 3 hairpin motifs, gray with 5 hairpin and 3 linear, green with 5 linear and 3 hairpin, and red with 5 and 3 linear. The gray had a frequency of 49.2% and the blue had a frequency of 25.8% Structures (1-120) 3 TAR PBS DIS (SL1) SD (SL2) Ψ (SL3)

9 but the motif only exists as a less fit component of an overall less fit structure. Therefore, the 5 poly(a) signal is dominantly occluded in a hairpin motif as indicated by experimental and computational results. On the other hand, the 3 poly(a) signal forms a less prevalent hairpin structure and a dominant linear structure. At lower populations of 4K, 8K, and 16K, the structures with the best energy exhibited a 3 poly(a) hairpin. As the population increased, the 3 poly(a) hairpin switched to a linear motif. Towards the higher populations (32K, 64K, 128K) in the final structures, the 3 poly(a) hairpin structure becomes either nonexistent or a less dominant structure. This trend is characteristic of all the nine tested sequences (data not shown). Analyzing the maturation of the virus in various individual MPGAfold runs revealed an oscillation between the 3 hairpin and linear poly(a) motifs. The variability of the 3 poly(a) signal between hairpin and linear states in the final structures and during maturation in the genetic algorithm may indicate the needed structural instability for polyadenylation as suggested in (21). Figure 3B depicts a structure with the dominant 5 and 3 poly(a) motifs (consensus structure). This particular structure exhibited a free energy that was slightly worse than the best overall structure (within 2%). In fact, the difference between the energies of the less fit and best fit structures was less than 5%. Since the 5 poly(a) signal and its surrounding domain are relatively stable in hairpin structures, the energy difference is mostly due to the rearrangement in the 3 end. This difference suggests that the 3 structure can easily switch conformations between the linear and hairpin poly(a) motifs while having a greater propensity for the linear association. 425 Structural Differentiation of the HIV-1 Poly(A) Signals The energetic stability of the entire HIV-1 construct also plays a role in the behavior of the poly(a) signal. In individual runs the structure of the virus appears to progress through different states: a multibranch structure to a linear structure with different poly(a) formations (Figure 4). In the final structures, the higher energy values of the structures were characterized by having both 5 and 3 poly(a) hairpins. The intermediate energy structures, which were the most populous, displayed a 5 hairpin and 3 linear poly(a) signal conformation (Figure 3B). For example, the LAI sequence exhibited the 5 hairpin and 3 linear structure 49.2% of the time and 25.8% for both the 5 and 3 hairpins. This trend seems to appear throughout the different constructs (Table II). In a few other cases, the structure transitions from a linear to a multibranch state. For instance, NDK, IBNG, and U455 showed a propensity for the 5 and 3 hairpin structure. These deviations appear to be a result of mutations in an asymmetric internal loop that is adjacent to the internal loop encompassing part of the 3 Poly(A) linear structure. For these three sequences, the structures with a 5 hairpin and 3 linear signal have a slightly worse overall energy than structures with the 5 and 3 hairpin. This delicate balance between the free energies shows potential significant 3 metastability of the poly(a) structure. In addition, NDK and IBNG have an unusual point mutation in the sequence that binds to the AAA portion of the signal. Instead of a UUU motif, the mutation creates a UGU for NDK and a UUC for IBNG that inhibits the effective formation of the 3 linear poly(a) structure (Figure 2B). To further analyze this issue, we utilized MPGAfold s sticky stem feature where a set of stems is biased to remain within a structure once they have naturally occurred through crossover or mutation. By stickifying the linearizing stems, the structure exposing the signal was able to form with overall structure energies that were approximately 5% less than the original folds, a relatively small energetic difference. Sequential Folding By using the sequential folding capability offered by MPGAfold, we were able to model the behavior of an elongating RNA sequence. Sequential folding emulates RNA transcription where the sequence is being folded as it forms. In this experiment, we ran the sequential folding algorithm that added one nucleotide per generation and then increased the addition of nucleotides to four. The addition of nucleotides at the end of each generation, whether by one, two, three, or four,

10 426 Gee et al. strengthened the formation of the 5 poly(a) hairpin in the 5 R to 3 U5 and the 5 R to 3 R HIV-1 LAI and CM240 sequences. The 5 poly(a) signal occurred in the final structures as a hairpin motif with a frequency of 100%. For the 3 side, the effects of sequential folding were quite contrary to that of the 5 end. The sequential folding of the LAI sequence did decrease the frequency of formation of the 3 poly(a) hairpin, causing it to appear only in less fit structures. As the number of nucleotides added per generation increased, the frequency of the 3 hairpin signal decreased further. Without sequential folding, the frequency of the 3 poly(a) hairpin was 40% over the span from 4K to 128K populations (120 final structures). When sequential folding was employed using one nucleotide per generation elongation, the frequency for the 3 hairpin structure across the 4K to 128K populations was approximately 27.5%. When the sequence was extended using two and three nucleotides per generation, the frequency of the hairpin decreased and reached a level of 23.3% for four nucleotides per generation. Similar results were achieved for the CM240 sequence where the frequency of the 3 poly(a) hairpin was 17.5% over the span from 4K to 128K populations with one and two nucleotide elongations and 14.2% with four nucleotide elongation. In the higher populations (64K and 128K) the 3 poly(a) hairpin was nonexistent. In addition, the 5 R to 3 R domains showed similar results where the frequency for the 3 poly(a) hairpin decreased as the elongation increased (data not shown). The sequential folding algorithm supports our hypothesis that while the 5 poly(a) hairpin structure is more dominant and stable, the 3 poly(a) region shows a different behavior than the 5 region by demonstrating variability in its structure. Discussion Polyadenylation is a process by which the 3 poly(a)-tail of most mrnas is created. The central element of polyadenylation is the poly(a) hexamer signal (AAUAAA), which exists in both the 5 and 3 untranslated regions (UTR). Previous studies have suggested that the 5 poly(a) signal of HIV-1 is sometimes occluded in a hairpin structure but may change conformations according to changes in the secondary structure (7-9). Through computational predictions, we suggest that the 5 and 3 poly(a) signals have dominant structural motifs but are also differentially metastable allowing the signal to be part of different structural conformations. Understanding the suppression of the 5 and activation of the 3 polyadenylation signals is essential for understanding the life-cycle of the virus. This extensive structural study reveals that the structural motif of the 3 poly(a) signal before and after the cleavage of the downstream 3 U5 region is dominantly a linear structure with a large asymmetric internal loop (Figure 2B). Partly bound in several weak A-U base pairs, the 3 poly(a) signal may be more amenable to the binding of CPSF and other protein factors because of its linear structure and its ability to transition between structural conformations. The dual and flexible conformations of the 3 hairpin and linear states within final and individual MPGAfold runs may indicate the lack of stability and induce the exposure needed for 3 polyadenylation, as indicated in (21). Past studies have shown in vitro and in vivo that an upstream enhancer element (USE) is essential for efficient polyadenylation (16, 17). The USE, located in the U3 region on only the 3 terminal (Figure 1), has been proposed to initially act as a binding or entry site for CPSF (18). In opposition, the suppressive hairpin structure, which is dominant and relatively stable on the 5 end in our tested constructs, may sequester the signal by disrupting protein complex formations. Studies have indicated that the hairpin structure has an inhibitory effect on polyadenylation (18, 21), specifically during the binding of the ternary RNA- CPSF-CStF complex to the poly(a) signal (18). By thermodynamically stabilizing the 5 poly(a) hairpin structure, the formation of the poly(a) complex was greatly reduced in HeLa nuclear cell extract with purified CPSF and CStF factors (18). In addition to the hairpin structure, complete repression of 5 polyadenylation may require the binding of the major SD site to the U1 snrnp in the HIV-1 leader region

11 (18, 19). In vitro studies have shown that destabilization of the 5 poly(a) hairpin triggers premature polyadenylation (20, 21), and an alternative structure to the suppressive 5 poly(a) hairpin is an internal loop (7-9). Thus, it can be anticipated that the 3 linear poly(a) structure, destabilized by an internal loop, or the variability between the 3 hairpin and linear motifs may trigger 3 polyadenylation. 427 Structural Differentiation of the HIV-1 Poly(A) Signals Studies in vitro on the 5 UTR reveal that the 5 poly(a) structure has the ability to exist in two different motifs, a linear long distance interaction (LDI) and a hairpin stem-loop (7, 8). The 5 poly(a) hairpin plays a key role in allowing dimerization by permitting the formation of the kissing loop hairpin interaction while the linear 5 poly(a) signal is detrimental to dimerization by binding to the DIS signal (7). However, studies have not addressed the functional and delicate balance between the dual conformations in both the structure of the 5 and 3 poly(a) signals. In this study, we have shown that there are two metastable but dominant poly(a) conformations that may determine alternate functional states of the HIV-1 viral RNA. The structures with both the 3 and 5 hairpins were, in general, less fit by approximately 2-5% than the structure with the best free energy, which exhibited linear conformations at both ends (Figure 4). Since the free energies are relatively comparable, the hairpin structures are able to exist in both 5 and 3 poly(a) conformations, as in the case of reverse transcription where the 5 and 3 poly(a) signals are in hairpin motifs that are neither weak nor excessively stable (37). Interestingly, the lowest energy structures had a conformation with both 3 and 5 linear poly(a) signals, though HIV-1 may have great difficulty reaching the low thermodynamic energy as reflected by the results of the 128K population run. Since in vitro lab studies have shown that a stable 3 poly(a) hairpin can efficiently inhibit polyadenylation (21), the fluctuation and delicate balance between the 3 hairpin and 3 linear motifs, as shown in our results, may serve as the unstable structure needed for polyadenylation to occur. Furthermore, the 5 poly(a) hairpin exhibits a potential tertiary interaction with the matrix coding region forming a long distance pseudoknot (35). This pseudoknot, which can exist only in the branched structures containing the 5 poly(a) hairpin loop, can inhibit translation of the gag gene (36). But, the linear 5 structure, which cannot support this pseudoknot interaction, may enhance translation. A recent ex vivo study has demonstrated that the branched multiple hairpin (BMH) structure on the 5 end is the dominant structure in cells (36). Though this supports our evidence for the dominance of the 5 poly(a) hairpin structure, it does not exclude the possibility that the LDI structure can form on either the 5 or 3 terminal, as demonstrated by in vitro studies. Computational and experimental results agree that both the 5 and 3 poly(a) signals are in metastable structures that can switch conformations to regulate viral function. Of course, one has to keep in mind that the simulations described in this paper are dependent upon approximate energy rules that are applied in a non-cellular environment. Dominance percentages should not be taken literally. However, based on past experience, MPGAfold seems to do a reasonable job of capturing important conformational states and folding pathways (9, 23-25). Though some experiments have been performed to try to identify the delicate balance between the two polyadenylation signals (21), the balance has not been described quantitatively and with structural details. It may be possible to verify the results of our computational experiments by making some of the constructs described and conducting various structural probing and/or gel shift experiments to determine the structural details and whether multiple populations of structures exist in proportions similar to what we have observed. Experimental determination whether, and, if so, in what percentage of cases premature polyadenylation versus correct polyadenylation occurs can also contribute to verification of our results. Acknowledgements We wish to thank the Advanced Biomedical Computing Center (ABCC) for their support. This work has been funded in whole or in part with Federal funds from the

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