SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA
© The Author(s) 2016
Received: 11 May 2016
Accepted: 23 October 2016
Published: 11 November 2016
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).
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.
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.
The retrovirus genome is a single-stranded positive sense RNA. The viral genome always occurs as a dimer in virus particles, and the interaction is non-covalent, since heating easily dissociates purified dimeric genomes into monomers. It has been suggested that the encapsidation and dimerization of retroviral RNA genome plays an important role at various stages of the viral life cycle, including virion formation, reverse transcription and recombination.
Electron microscopy studies have suggested that the 5′ region of both viral RNAs contains a primary contact point and is referred to as the dimer linkage structures (DLS) [1–3]. The presumptive primary DLS of HIV-1 has been mapped to a region near the major splice donor and biochemical analysis indicates that the DLS probably consists of multiple stem-loop structures . Within the DLS, stem-loop 1 (SL1) has been regarded as the most important region, which forms a stem-loop structure with a hairpin loop containing a six-nucleotide palindromic sequence . The dimer formation would occur through a kissing hairpin mechanism by which the two RNAs would form an initial loop–loop contact based on complementary anti-parallel base pairing at the SL1 loops . So far, several structural models of psi or DLS such as “seven stem-loops” , “branched multiple hairpin”  or “pseudoknot-like shape”  have been proposed. As SL1 is expected to be a highly stable structure, computer predictions have indicated the shapes of SL1 within the models are all rather similar. The reports performing chemical probing and mapping of viral RNA including SHAPE assay (selective 2′-hydroxyl acylation analyzed by primer extension) have proposed several total secondary structure models of HIV-1 psi [9–12]. Moreover, the standalone structure of SL1 and whole structure of HIV-1 psi have been solved and characterized by NMR to some extent [13–16]. However, there have been few attempts to pursue the precise functional structure of SL1 in the context of a live virus and thus the role of SL1 structure within the overall HIV-1 DLS in vivo is still not completely understood.
We previously developed a unique system to assess DLS operation within the HIV-1 virion and identified the region which is necessary and sufficient for HIV-1 genome dimerization within the virion . In addition, we constructed a new packaging assay system based on the real-time RT-PCR with multiple probes. This system enabled a simple, reliable and reproducible quantification of HIV-1 genome-packaging efficiencies. Using these systems coupled with loss- and gain-of-function studies using site-directed mutagenesis, we performed the mapping of the functional base pairs within SL1 required for HIV-1 genome dimerization, packaging and replication, in much more further detail. Interestingly, the study revealed some unique information that has been never suggested previously. Based on these data, we made a modified SL1 structural model and also propose new possible processes of the HIV-1 RNA genome dimer formation. The importance of certain bases within SL1 is also discussed.
The replication-competent HIV-1 proviral clone pNL4-3 (subtype B) , its Env mutant pNLNh , or its derivative DDNLp4∆2 (renamed as DDNd2)  were used as progenitors for the mutant constructs described below. Mutant plasmids were constructed with standard methods.
293T cells  (approximately 3 × 106) were seeded on dishes (diameter, 100 mm) the day before transfection with plasmid DNA (5 µg) by means of the calcium phosphate precipitation method . The day after transfection, the supernatant was replaced with fresh medium.
At 48–72 h post-transfection, the media was centrifuged and supernatant was used for infection into MT-4 cells. Equal amounts of p24 were inoculated into the cells. For general experiments, 1 × 105 MT-4 cells were infected with 20 ng-p24 amount of virus. For “low-moi” experiments, 1 × 105 cells were infected with 4 ng-p24 amount of virus. The supernatants of the MT-4 were harvested every two to four days for multiple replication assays. Five microliters of each cell supernatant was subjected to exogenous RT assay as described previously .
Isolation of RNA from virions and cells
At 48–72 h post-transfection, virus particles were collected from the media as described elsewhere . The physical virus titer was determined with an ELISA assay kit for quantitation of CA-p24 (ZeptoMetrix, Inc., Buffalo, NY). To isolate RNA from particles, virions were disrupted by the addition of 1% sodium dodecyl sulfate (SDS) and treated with proteinase K (300 µg/ml) at room temperature for 60 min, followed by TE-saturated phenol/chloroform extraction, chloroform extraction, and ethanol precipitation.
Real-time RT-PCR based packaging assay
To examine the accurate genome packaging efficiency of HIV, a real-time RT-PCR based packaging assay was developed. Basic concept is similar to an RNase protection assay with wild-type control . To differentiate the control viral genome from those of the mutants of interest, a series of silent mutations for eight amino acids within CA region of pNLNh plasmid  were introduced and named as pNLNh-CAmut. This mutation did not affect virion production or genome packaging ability (data not shown.). A pair of primers to amplify the CA region (5′-ATCAAGCAGCCATGCAAATGTT-3′: FWdrosten and 5′-TGAAGGGTACTAGTAGTTCCTGCTATGTC-3′: RVdrosten) and two dual-labeled probes designed to anneal the CA region of either pNL4-3 or CAmut (5′CAL Fluor Red610-accatcaatgaggaagctgcagaatggga-3′BHQ-2: CAdrostenWT or 5′FAM-acTatTaaCgaAgaGgcAgcTgaGtggga-3′BHQ-1: CAdrostenMut, amino acid seq: TINEEAAEW) were synthesized (Biosearch Technologies, Inc. Petaluma, CA). A series of SL1 point mutants were each separately co-transfected into 293T cells along with pNLNh-CAmut as a control viral expression construct. Viral and cytoplasmic RNA isolated from the transfectant were applied for real-time RT-PCR with the primers and the probes. The PCR was performed with One Step PrimeScript RT-PCR kit and Thermal Cycler Dice (Takara Bio, Ohtsu, Japan). By comparing the amount of the control and the mutant RNA in virus and cell, it was possible to determine the effect of each of the mutations on relative encapsidation efficiency. Relative encapsidation efficiencies were calculated as the ratio of the amount of mutant to control (pNLNh-CAmut) genomic RNAs in the virions, with normalization to the cytoplasmic levels of the two RNAs.
Northern blotting analysis
W represents ratios of monomer content to total RNA of virions at room temperature produced from DDNd2 (WT), which contains two wild-type DLSs on one viral genome. M represents that from each DDNd2 derivative mutant construct (generally carrying the mutation at the original position of DLS: 5′ side). B represents that from NLNh (Neg.) (Additional file 1: Figure S1). The D value of the Neg. is zero and that of WT is one in each experiment. When the combination of mutants severely affects dimer formation, the monomeric genome content of the virion is reduced and D becomes close to zero. Thus, the D value was expected to represent the magnitude of the effect of the DLS mutation in virions.
HIV-1 genome recombination assay was performed as described in our previous papers [26, 27]. In short, two similar vectors with the same or different dimerization signals (DLS) were constructed (Additional file 1: Figure S2A) and co-transfected together with or without the VSV-G expression vector (pCG-VSVG) (Additional file 1: Figure S2B). Vector A carries a surface biomarker (Mark A: murine HSA) and an inactivated eGFP gene with amino-terminal mutation (GFP∆N), while vector B carries another biomarker (Mark B: murine CD52) and an inactivated eGFP gene with carboxyl-terminal mutation (GFP∆C). After transfection, the released virions can be expected to co-package the homo- or hetero-dimerized vector genome, while the ratio of homo- to hetero-dimerization should be one-to-one if the genome expression efficiency of the two vectors is similar (Additional file 1: Fig. S2C). Without VSV-G, the expression of HSA and CD52 indicates transfection efficiency of the vectors, and no eGFP expression should be observed. With VSV-G expression, pseudotyped virions have been observed to cause retro-transduction to the producer cells  and the number of marker genes expressing cells in the transfectant increases with an increase in the occurrence of retro-transduction. Recombination of the two vectors can be assumed to occur only in retro-transduced cells, and is monitored in terms of further restoration and expression of the eGFP gene (Additional file 1: Figure S2C). Transfection and retro-transduction efficiency are measured by expression of Mark-A and -B, while the transduction efficiency is estimated by subtracting marker gene expression ratios of the VSV-G negative sample (bA, bB%) from those of the positive sample (rA, rB%). Finally, the recombination efficiency is estimated by calculating the ratio of eGFP expressing cells (rG–bG%) in vector-transduced cells. If we assume that one of the recombination events always occurs between the ∆N and ∆C mutation of eGFPs during reverse transcription, 50% of the recombination events should result in reconstitution of the eGFP gene. In addition, 50% of the virions from doubly transfected cells possess a hetero-dimerized genome, and thus have the potential to reconstitute eGFP. This means that a ratio of eGFP positive cells of 25% in Mark-A or -B positive cells should be the maximum value for recombination. We therefore adopted this maximum ratio for easy indexing by quadrupling the numeric results (Additional file 1: Figure S2C).
Construction of three-dimensional (3D) structural model of the SL1
Determination of the “bubble” shape and its contributing nucleotide bases: Which is the actual in vivo form?
Contribution of G–C pairs in the upper stem
Overall in the upper stem, G251–C267 and C252–G266 basepairings appeared to occur in vivo, while on the other hand, the basepairing of G254–C264 might be dispensable for viral activity.
Contribution of G–C pairs in lower stem
Effect of hairpin loop modification to disrupt kissing-loop interaction
There are many reports concerning HIV-1 psi RNA structure employing various technologies nowadays [9–16]. Although such technologies have given us notably information, they have limitations for complete realization of the structure within virion or cell. For example, the primary information provided by RNA mapping with chemical probing is only that which nucleotides form double strand or not. Solving molecular structure by crystallography or NMR is performed in in vitro condition, which might not fully reflect them in native status. Thus even simple and ordinary methods, precise mutagenesis of psi RNA and analysis on viral functions of the mutants are still significant actions for understanding an overall structure of psi RNA in virion. In this report, we successfully analyzed precise roles of G–C pairings within the HIV-1 SL1 region and found several possibilities of novel genomic structures, which may have functional merits.
In the bubble region, we confirmed that the base pairing of C248–G270 was very important for viral activity and for model A (Fig. 1b) which appeared to be the functional structure of SL1. Contrary to the suggestions from computer predictions, SL1 forms the bubble with 3–1 bases (Fig. 1b: model A) in vivo, despite showing slightly lower stability. We were also interested in the non-basepairing guanines at the bubble. We constructed and examined two mutants, which were forced to form a base-pair (G247C and G273C) and a mutant which possesses cytidines instead of guanines (G247C/G273C) at the bubble. As a result, none of the mutants retained full viral activity, suggesting that the exposed guanines at the bubble are important for both dimerization and viral replication. As several reports have implicated [10, 34–38], the preference manifested by NC for unpaired guanine bases may contribute to the RNA-NC interaction at the beginning of dimerization and the formation of the model A structure.
Upon investigating the upper and lower stems, most of the G–C pairings in SL1 stem appeared to be important for SL1 function as expected (Figs. 3, 4). Surprisingly, one pairing proximal to the hairpin loop (G254–C264) seemed to be tolerable for base substitution or dispensable (Fig. 3). As this G–C interaction limits the size of the loop, this data might suggest that the putative loop is larger than initially believed, composed by eleven exposed nucleotides. Since the six-nucleotide palindrome must be aligned antiparallelly for the initiation of dimer formation, the flexibility of the hairpin loop attributed by these non-pairing bases should yield some advantages to achieve kissing-loop formation.
Additional unexpected results were obtained from one basepair at the bottom of the lower stem (C243–G277). The exchange of these bases (C243G–G277C) did not affect viral replication and genome packaging, but the ability of the mutated DLS to interact with wild-type DLS was notably reduced (Fig. 4). The following experiment revealed that upon concordance of intermolecular basepairing (GG–CC, CC–GG, or GC–GC) of this position showed some restoration of dimerization efficiency compared to those of the discordant pairs (Figs. 4d, 5b). Especially, homo-dimerization of the double mutant DLS (GC–GC) was sufficiently comparable to that of the wild-type. In addition, the genome recombination rate between homomeric double mutants was also similar to that of the wild type (Fig. 5c). From these data, we consider that the bottom portion of SL1 might operate at an alternative dimer interface—at least transiently—during some steps of genome dimerization. There was a discrepancy also between replication and dimerization of the mutants. The mutant C243G showed much less dimerization ability compared to that of G277C whereas viral replication of C243G was better than that of G277C (Fig. 4b, d). We think there are two possibilities. One is that the packaging efficiency (C243G > G277C) is more important than the dimerization ability (C243G < G277C) for replication of the mutants in this case. The other is, which is most likely, that the results of dimerization efficiency of the mutants concerning C243–G277 in Fig. 4d might not reflect the actual dimerization ability of them in virion. As explained in the “Results” section, our dimerization assay system measures the hetero-dimerization between a mutant and the wild-type DLS (Fig. 5a, Additional file 1: Figure S1C). As described in Fig. 5b, homo-dimerization ability of C243G/G277C mutant is far different from the result in Fig. 4d. Thus, although we do not have the data, it is possible that homo-dimerization efficiency of C243G single mutant genome in virion is not as low as the data shown in Fig. 4d.
In the upper or lower stem, all mutants having exchanged base pairs (G–C to C–G or vise versa) did not alter the viral activity much. This suggests that base pair formation is important for SL1 function, but the specific base location is not, with the exception of the bubble guanines.
Complete base pairing between two hairpin loops of SL1 appeared to be important for efficient viral replication (Fig. 6). Co-existence of 6Gs and 6Cs genome in virion restored viral replication and dimerization (Fig. 6b, c). These data suggested that the virion preferably packages hetero-dimerized genome with complete basepairing in this case and thus efficient genome packaging helped viral replication. Although it is consistent with the currently-accepted theory , evidence of some replicative ability was observed in 6Gs and 6Cs mutants, albeit it was reduced and delayed. As the necessity of DIS loop for virus replication was previously discussed in some reports [40, 41], this might suggest that kissing loop interaction is not absolutely required for virus replication or that the kissing loop is somehow formed even between non-complimentary strands to establish a functional psi in a certain condition.
We performed a series of precise, functional mapping studies of SL1 in HIV-1 DLS and discovered several possibilities for a novel structure. In the newly proposed structure, the hairpin loop of SL1 appeared larger, and DIntS may mediate a more complicated mechanism of genome dimerization than previously understood. A recent report suggested that HIV-1 Gag precursor Pr55 preferentially binds to the free guanines at the bubble and at the lower stem of SL1 . These data are consistent with our own results and also imply the possibility of important roles of the lower stem of SL1 on intermolecular interactions. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.
SS and JS performed the experiments, analyzed the data, and generated the text and the figures. MY and HS performed computer analysis and generated the text and the figures. JS and TS contributed to drafting of the manuscript, and supervised the study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Research Program on HIV/AIDS from Japan Agency for Medical Research and development, AMED.
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