Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging
© Das et al.; licensee BioMed Central Ltd. 2012
Received: 27 March 2012
Accepted: 4 July 2012
Published: 24 July 2012
The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA.
We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging.
These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.
In vitro studies demonstrated that the HIV-1 leader RNA cannot only fold the branched multiple hairpin (BMH) conformation, but also an alternative conformation in which DIS sequences interact with the polyA region [20–22]. This long-distance interaction (LDI) prevents exposure of the DIS element and can thus control the formation of RNA dimers [20, 23, 24]. More recently, an alternative LDI structure was proposed in which the DIS element interacts with U5 sequences downstream of the polyA hairpin . This U5-DIS interaction similarly occludes the DIS loop sequence and prevents RNA dimerization. The BMH and LDI conformers may provide the virus with a riboswitch that coordinates leader RNA functions like dimerization, packaging and translation. Although the TAR hairpin is present in both leader RNA conformations, it has previously been suggested that mutations in TAR can affect the LDI-BMH riboswitch and consequently several leader RNA functions . Indeed, TAR has been shown to influence dimerization of the viral RNA genome [26–28], packaging of the genomic RNA into virions [10, 12–14, 21, 29, 30] and the strand transfer step of reverse transcription [31–34]. Previous attempts to dissect the functions of TAR in vivo, i.e. in the context of the replicating virus, were hampered because mutations in this element cause a severe transcription and replication defect [10, 29, 35]. We created an HIV-1 variant with an alternative transcription axis and demonstrated that the TAR hairpin can be truncated and even completely deleted when not required for Tat-mediated activation of transcription . Surprisingly, virus variants in which the TAR sequence was only partially deleted exhibited a severe replication defect. We showed that opening of the 5′ TAR element caused aberrant folding of the leader RNA in vitro, resulting in uncontrolled dimerization of leader RNA transcripts due to induced exposure of the DIS hairpin . In this study, we used the same set of TAR mutants to demonstrate that the processes of genomic RNA dimerization and packaging in virus particles are seriously affected by misfolding of the leader RNA.
We previously designed an HIV-1 variant in which the Tat-TAR transcription mechanism was inactivated through mutation and functionally replaced by the doxycycline (dox)-inducible Tet-On gene regulation system (Figure 1A). This HIV-rtTA variant replicates exclusively in the presence of dox and does not require TAR for the activation of transcription. To study additional functions of the TAR hairpin in HIV replication, we generated HIV-rtTA variants in which the left or the right side of the TAR stem was partially deleted (A and B mutants in Figure 1B, respectively). These deletions open the TAR stem and thus destabilize this RNA element. The A and B deletions were combined in the AB double mutant, which resulted in truncation of the TAR hairpin. Whereas the A and B virus mutants showed a severe replication defect, the AB mutant replicated as efficiently as the original HIV-rtTA virus . Structure probing of in vitro produced leader RNA transcripts demonstrated that opening of the 5′ TAR hairpin resulted in stabilization of the polyA hairpin, which forced the leader RNA in the BMH conformation . We here set out to analyze which step of the HIV-1 replication cycle is affected by this mutation-induced conformational change of the leader RNA.
We first examined the effect of the TAR mutations on the production of HIV-1 RNA and its packaging into virions. C33A cervix carcinoma cells were transfected with the HIV-rtTA molecular clones that contain either the wild-type or modified TARm sequence in both LTRs (A5+3, B5+3 and AB5+3) and cultured with dox for two days. Because we previously observed that opening of the TAR hairpin at the 3′ end of the viral genome reduced the polyadenylation efficiency of the viral transcripts , all molecular clones contained an SV40-derived polyadenylation signal downstream of the viral genome. Transcripts that are not polyadenylated at the 3′ LTR site will be polyadenylated at this SV40 site, resulting in a 276-nt extension .
We similarly compared packaging of the A5 RNAs (with an opened TAR hairpin structure) and AB5 RNAs (with a truncated but stable hairpin structure) by co-transfecting cells with both HIV-rtTA mutants (A5/AB5 lanes in Figure 4B). For the cDNA/PCR analysis of the RNAs present in the cells and virions, we again used primers that resulted in differently sized products for the A and AB variants (AB products being 10 nucleotides smaller than the A products). Analysis of the intracellular RNA from the A5/AB5 transfected cells revealed a similar production of unspliced and spliced transcripts from the A5 and AB5 constructs (Figure 4B, left panels). Analysis of the unspliced and spliced RNAs in the A5/AB5 virus particles revealed the dominant presence of AB5 transcripts (Figure 4B, right panels). These results demonstrate that the AB5 mutated unspliced and spliced transcripts are more efficiently packaged than the corresponding A5 mutated RNAs. In both packaging competitions (TARm/B and A/AB) we thus observed that opening of the 5′ TAR structure element does not only reduce the packaging efficiency of the unspliced RNA but also that of the spliced viral RNAs, which argues against a direct positive effect of TAR opening on packaging of spliced RNAs. It thus seems likely that the increased level of spliced RNAs observed in the A5 and B5 mutated virions (Figure 3) was an indirect effect of the reduced packaging of unspliced transcripts.
This study is based on the surprising finding that the HIV-rtTA variant, which does not need the 5′ TAR RNA element for Tat-mediated activation of transcription, is severely replication impaired by a deletion in the left or right side of the TAR stem that opens the hairpin structure (A and B mutants), but not by the combined deletion that truncates this stem-loop element (AB). We demonstrate that introduction of the A and B deletions in the 5′ TAR element (A5 and B5 mutants) does not influence the production and splicing of viral RNA, which is in agreement with previous observations . However, these TAR-opening mutations affected the configuration of the unspliced viral RNAs in cells (more monomeric RNAs, less large RNA complexes) and caused reduced packaging and dimerization of these RNAs in virions. The reduced level of unspliced RNA in virions coincided with an increased level of the spliced viral RNAs. In contrast, unspliced viral RNAs with a truncated TAR hairpin (AB5) exhibited the wild-type RNA pattern in cells and normal packaging and dimerization in virions. The observed changes in RNA configuration, dimerization and packaging thus correlate with the virus replication phenotype.
The unspliced HIV-1 transcript is used as mRNA for the synthesis of the Gag and Pol proteins and as genomic RNA that is encapsidated and present as dimer in the viral particle. During this packaging process, the genomic RNA must be selected from a multitude of cellular and spliced viral RNAs. This selection involves cis-acting RNA elements and the trans-acting nucleocapsid (NC) domain of the Gag protein . The major packaging signal ψ is positioned downstream of the splice donor site and is thus exclusively present in the unspliced transcripts . In addition to the ψ motif, other leader RNA signals upstream and downstream of the SD site have been implicated in the process of RNA packaging [2, 10, 13, 14, 16, 28, 39, 42]. The upstream elements (including TAR, polyA, PBS and DIS) are present in both the unspliced and spliced transcripts, suggesting that these elements cannot contribute to the packaging specificity. However, although the spliced HIV RNAs are packaged much less efficiently than the unspliced RNA, they are packaged more efficiently than cellular RNAs , which argues for a contribution of the upstream region. Opening of the 5′ TAR structure reduced packaging of the unspliced RNA and increased packaging of spliced RNAs. These results suggest that the spliced and unspliced viral RNAs compete for packaging, which is in agreement with previous observations [36, 37, 39]. We demonstrated that the opened TAR structure did not selectively improve packaging of the spliced transcripts, but rather reduced the packaging efficiency of both unspliced and spliced RNAs. The relative amount of spliced transcripts increased, which indicates that packaging of the unspliced transcripts was more seriously affected.
We not only observed genomic RNA monomers and dimers when analyzing the RNA content of cells and virions, but also larger RNA complexes (Figure 5). The nature of these complexes remains unclear. Possibly, the complexes are formed through interactions between multiple unspliced viral RNAs or through interactions between the unspliced RNAs and spliced viral RNAs or cellular RNAs. We, however, cannot exclude that they are formed during or after cellular and virion RNA extraction. Whereas the unspliced RNA molecules were predominantly present as RNA dimer in TARm and AB5 virions, these RNAs were largely present as complexes in the corresponding cells. The A5 and B5 mutations resulted in less intracellular RNA complexes and more monomeric RNA. Whereas the intracellular level of RNA dimers was seemingly unaffected by the A5 and B5 mutations, the RNA dimer level in the corresponding virions was found to be reduced. Although it has been observed that dimerization and packaging can be dissociated [43–46], a coupling of RNA dimerization and subsequent packaging has been suggested by other studies [21, 47–53]. If dimerization is a prerequisite for packaging, our results would suggest that dimeric RNA molecules with a complete or truncated 5’ TAR element (TARm and AB5, respectively) are more efficiently packaged in virus particles than dimeric RNA molecules with an opened 5’ TAR structure (A5 and B5). On the other hand, the direct correlation that we observe between the intracellular complex level and the RNA dimer content of the corresponding virions (TARm and AB5: high complex level in cells and high dimer level in virions; A5 and B5: low complex level in cells and low dimer content of virions) may suggest that the RNA complexes are the actual targets for packaging, which are later transformed into dimers. However, we have not verified the configuration of the RNA molecules in newly released virions, and it has been shown that RNA monomers (or RNAs that appear as monomers when assessed by gel electrophoresis) can also be efficiently packaged and subsequently mature into dimers [43, 46].
Previous in vitro RNA structure probing experiments indicated that opening of the TAR structure resulted in an interaction between unpaired TAR nucleotides and downstream U5 sequences, which caused extension of the adjacent polyA hairpin . Stabilization of this hairpin results in occlusion of the polyadenylation signal and reduced polyadenylation at the 3′ end of the viral transcripts [5, 6, 17, 54]. An altered RNA conformation may also explain the deleterious effect of TAR opening in the 5′ leader RNA. Extension of the polyA hairpin with TAR and U5 nucleotides will destabilize the U5-AUG duplex that is formed immediately downstream of the polyA hairpin. It has previously been shown that such destabilization reduces dimerization of the viral RNAs . Alternatively, TAR opening and subsequent polyA hairpin extension may affect dimerization more indirectly by influencing exposure of the DIS hairpin. Whereas the wild-type leader transcript preferentially adopts an LDI conformation in which the DIS signal is occluded through either a polyA-DIS or U5-DIS interaction [20–25], extension of the polyA hairpin will counteract this interaction and shift the RNA toward the alternative BMH conformation in which the DIS sequence is exposed . Ooms et al. previously showed that a change in the LDI/BMH equilibrium correlates with reduced packaging of the genomic RNA into virus particles. In agreement with this, we observed that the A5 and B5 mutations do indeed reduce packaging of the unspliced RNA. Moreover, we previously measured aberrant dimerization of TAR-mutated leader RNA molecules in in vitro assays. The A and B leader RNAs were found to dimerize more efficiently than the wild-type and AB molecules, which may be due to the increased exposure of the DIS signal in these short RNAs . The current in vivo analysis confirms that the A5 and B5 mutations affect the dimerization process, but in this case a more complex pattern is observed. The mutations did not seemingly affect the intracellular RNA dimer level, but reduced complex formation and increased the monomeric RNA level in cells. Moreover, these TAR opening mutations reduced the RNA dimer content of the virions. These results may seem contradictory, but we note that there is good evidence that in vitro RNA dimerization only partially mimics the in vivo dimerization process. For instance, the DIS drives in vitro RNA dimerization via a loop-loop kissing interaction, but a 4-nt deletion in the loop-exposed palindrome surprisingly does not prevent HIV-1 replication and does not affect the stability of the in vivo dimer . This and other results [55, 56] indicate that retroviral RNA dimerization is a far more complicated process than simple DIS loop kissing, and up-regulation of what is perhaps the initial step in the RNA dimerization process may be detrimental to the complete cascade of RNA dimerization.
We previously showed that HIV-rtTA and a similarly designed dox-controlled SIV-rtTA variant replicate efficiently upon complete deletion of the TAR hairpin [30, 57]. These results indicate that TAR has no essential function in the HIV and SIV life cycle other than its role in Tat-mediated activation of transcription. In agreement with this, we observed efficient packaging of HIV-rtTA genomic RNAs in which TAR was truncated (this study), completely deleted or replaced by a non-related hairpin (ER2 and ER3 variants described in ; results not shown). Moreover, Heng et al. very recently demonstrated that TAR deletion did not significantly affect RNA dimerization, NC binding and RNA packaging . However, as we show in this study, opening of the 5′ TAR hairpin perturbs the leader RNA structure and affects both dimerization and packaging of the viral RNAs. Furthermore, we earlier demonstrated that opening of the 3′ TAR structure results in stabilization of the adjacent polyA hairpin that masks the polyadenylation signal, causing inhibition of polyadenylation . Our combined studies thus demonstrate that although the TAR hairpin is not required for RNA packaging, dimerization and polyadenylation, mutations in TAR can affect these processes in an indirect way by disturbing the structure at the 5′ and 3′ end of the viral RNA. Apparently, the wild-type TAR hairpin is sufficiently stable to prevent detrimental interactions with other RNA domains. This insight explains the strong evolutionary pressure to close disrupted TAR hairpins in virus evolution experiments [19, 30, 59, 60]. These studies illustrate how mutations that are designed to explore the function of a specific RNA element can affect viral replication in an indirect way through unforeseen conformational perturbation of the viral RNA genome.
HIV-1 does not require TAR for dimerization and packaging of its RNA genome, but mutations in TAR can affect these processes in an indirect way. We demonstrate that opening of the TAR hairpin structure alters the leader RNA conformation, which reduces packaging and dimerization of the unspliced viral RNAs.
Cells and viruses
C33A cervix carcinoma cells (ATCC HTB31)  were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and minimal essential medium nonessential amino acids, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C and 5% CO2. Construction of the infectious HIV-rtTA molecular clone and variants with a deletion in the 5′, the 3′ or both TAR elements was described previously [30, 62]. In all constructs used in this study an SV40 polyadenylation site is positioned downstream of the viral genome as described previously .
C33A cells were cultured to 60% confluency in 3 ml complete medium in 10-cm2 wells and transfected with 5 μg HIV-rtTA plasmid by calcium phosphate precipitation as previously described . Cells were cultured in the presence of 1 μg/ml doxycycline (dox) (Sigma D-9891), and the culture medium was changed after 16 h. The virus level in the culture medium was quantitated at 2 days after transfection by CA-p24 enzyme-linked immunosorbent assay (ELISA) . RNA was isolated from the culture supernatant by the method of Boom et al.. The cells were washed with phosphate buffered saline (PBS), briefly incubated with 0.5 ml 0.05% trypsin-EDTA (Invitrogen) till they detached and resuspended in 1 ml 10% FCS-containing medium to inactivate trypsin. Cells were subsequently centrifuged at 2,750 g for 5 min, washed in 1 ml PBS, centrifuged at 2,750 g for 5 minutes, resuspended in 0.6 ml RLT buffer (QIAGEN) and homogenized with a QIAshredder column (QIAGEN). Total RNA was isolated with an RNeasy kit (QIAGEN) and contaminating DNA was removed with RNase-free DNase (QIAGEN) during isolation.
For the isolation of RNA from purified virions, C33A cells were cultured in 20 ml complete medium in 75-cm2 plates to 60% confluency, transfected with 40 μg HIV-rtTA and cultured with 1 μg/ml dox. The culture supernatant was harvested 2 days after transfection and cells were removed by low-speed centrifugation (10 min at 1500 rpm). The supernatant was filtered through a 0.45-μm filter and virion particles were pelleted by centrifugation at 32,000 rpm (175,000 g) for 90 min at 4°C in a Beckman SW32 Ti rotor. The virions were resuspended in 0.6 ml RLT buffer. The RNA was isolated as described above and resuspended in 50 μl water.
Northern blot analysis
For denaturing Northern blot analysis, 5 μg cell-derived RNA in 10 μl water or 10 μl virion-derived RNA were mixed with 10 μl denaturing sample buffer (80 mM MOPS pH 7.0, 20 mM sodium acetate, 14% formaldehyde, 0.1 mg/ml ethidium bromide, 1 mg/ml orange G, 13 g/ml sucrose), heated at 65°C for 10 min and subsequently electrophoresed on a 1% agarose gel in MOPS buffer (40 mM MOPS, 10 mM sodium acetate, pH 7.0) with 7% formaldehyde at 100 V for 4 h. For non-denaturing Northern blot analysis, 10 μl RNA was mixed with 5 μl sample buffer (30% glycerol and 0.25% bromophenol blue), incubated for 10 min at 37°C (or 55°C when indicated) and electrophoresed on a 0.9% agarose gel in 1 x Tris-Borate-EDTA buffer at 100 V for 4 h. The RNA was subsequently denatured by soaking the gel in 3 volumes of 10% formaldehyde at 65°C for 30 min.
The RNA was transferred onto a positively charged nylon membrane (Boehringer Mannheim) with 20 x SSC by means of capillary force for 16 h. The RNA was linked to the membrane using a UV crosslinker (Stratagene). A 32P-labeled probe corresponding to the nearly complete HIV-rtTA genome was generated by random-primed labeling (High Prime DNA Labeling kit; Roche Diagnostics) of the 3.9 and 5.5 kb SalI fragments derived from the HIV-rtTA plasmid. Prehybridization and hybridization was done in ULTRAhyb buffer (Ambion) at 55°C for 1 and 16 hours, respectively. The membrane was then washed two times at room temperature for 5 min with low-stringency buffer (2 × SSC, 0.2% SDS) and two times for 10 min at 50°C in high stringency buffer (0.1 × SSC, 0.2% SDS). Images were obtained using the PhosphorImager (Amersham Biosciences) and data analysis was performed with the ImageQuant software package.
RT/PCR analysis of viral RNA
RNA was reverse transcribed with ThermoScript reverse transcriptase at 50°C (Invitrogen) using the supplied oligo(dT)25 and random hexamers primers. The cDNA product was used as template in a PCR assay with primers 1 (GAG ACC ATC AAT GAG GAA GCT GCA GAA TGG GAT; position +942 /+974 with +1 as the transcription start site) and 2 (GGC CGG CCC TTG TAG GCC GGC CAG ATC TTC CC; +1663/+1638) to detect unspliced RNA, with primers 3 (TCA ATA AAG CTT GCC TTG AGT GC; +71/+93) and 4 (CTA TGA TTA CTA TGG ACC ACA CA; +5724/+5702) to detect the single spliced RNA, and with primers 3 and 5 (CTC CGC AGA TCG TCC CAG AT; +8102/+8083) to detect the double spliced RNA. The cDNA was denatured at 94°C for 5 min and PCR-amplified in 30 cycles of 1 minutes 95°C, 1 minutes 55°C, 2 minutes 72°C and a final extension time of 7 minutes at 72°C. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide. In the co-transfection experiment, the unspliced transcript was detected with primers 1wt/B (TAA TAC GAC TCA CTA TAG GTC TCT CTG GTT AGA CCA G; HIV-rtTA positions +1/+20 underlined) or 1A/AB (CTA ATA CGA CTC AGT ATA GGG TCT CTC TG-Δ11/24-GAGC ATT GGA; HIV-rtTA positions +1/+34 underlined) plus 2+348 (CAT CGA TCT AAT TCT CCC CCG CTT AAT ACT GAC GC; +382/+348), and the splice products were detected with primers 1wt/B or 1A/AB plus 4 (single spliced) or 5 (double spliced). The PCR products were digested with NarI before gel analysis.
This research was sponsored by the Netherlands Organisation for Scientific Research (Chemical Sciences Division; NWO-CW; Top grant) and the Aids Fonds (grant 2005022).
- Berkhout B: Structure and function of the human immunodeficiency virus leader RNA. Prog Nucleic Acid Res Mol Biol. 1996, 54: 1-34.View ArticlePubMedGoogle Scholar
- Lu K, Heng X, Summers MF: Structural determinants and mechanism of HIV-1 genome packaging. J Mol Biol. 2011, 410: 609-633. 10.1016/j.jmb.2011.04.029.PubMed CentralView ArticlePubMedGoogle Scholar
- Richter S, Ping YH, Rana TM: TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication. Proc Natl Acad Sci USA. 2002, 99: 7928-7933. 10.1073/pnas.122119999.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei P, Garber ME, Fang S-M, Fisher WH, Jones KA: A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell. 1998, 92: 451-462. 10.1016/S0092-8674(00)80939-3.View ArticlePubMedGoogle Scholar
- Klasens BIF, Das AT, Berkhout B: Inhibition of polyadenylation by stable RNA secondary structure. Nucleic Acids Res. 1998, 26: 1870-1876. 10.1093/nar/26.8.1870.PubMed CentralView ArticlePubMedGoogle Scholar
- Klasens BIF, Thiesen M, Virtanen A, Berkhout B: The ability of the HIV-1 AAUAAA signal to bind polyadenylation factors is controlled by local RNA structure. Nucleic Acids Res. 1999, 27: 446-454. 10.1093/nar/27.2.446.PubMed CentralView ArticlePubMedGoogle Scholar
- Beerens N, Groot F, Berkhout B: Initiation of HIV-1 reverse transcription is regulated by a primer activation signal. J Biol Chem. 2001, 276: 31247-31256. 10.1074/jbc.M102441200.View ArticlePubMedGoogle Scholar
- Beerens N, Berkhout B: The tRNA primer activation signal in the HIV-1 genome is important for initiation and processive elongation of reverse transcription. J Virol. 2002, 76: 2329-2339. 10.1128/jvi.76.5.2329-2339.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Abbink TE, Berkhout B: RNA structure modulates splicing efficiency at the HIV-1 major splice donor. J Virol. 2007, 82: 3090-3098.PubMed CentralView ArticlePubMedGoogle Scholar
- Das AT, Klaver B, Berkhout B: The 5′ and 3′ TAR elements of the human immunodeficiency virus exert effects at several points in the virus life cycle. J Virol. 1998, 72: 9217-9223.PubMed CentralPubMedGoogle Scholar
- Luban J, Goff SP: Mutational analysis of cis-acting packaging signals in human immunodeficiency type 1 RNA. J Virol. 1994, 68: 3784-3793.PubMed CentralPubMedGoogle Scholar
- McBride MS, Schwartz MD, Panganiban AT: Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J Virol. 1997, 71: 4544-4554.PubMed CentralPubMedGoogle Scholar
- Clever JL, Miranda D, Parslow TG: RNA structure and packaging signals in the 5′ leader region of the human immunodeficiency virus type 1 genome. J Virol. 2002, 76: 12381-12387. 10.1128/JVI.76.23.12381-12387.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Helga-Maria C, Hammarskjold ML, Rekosh D: An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA. J Virol. 1999, 73: 4127-4135.PubMed CentralPubMedGoogle Scholar
- Russell RS, Hu J, Laughrea M, Wainberg MA, Liang C: Deficient dimerization of human immunodeficiency virus type 1 RNA caused by mutations of the u5 RNA sequences. Virol. 2002, 303: 152-163. 10.1006/viro.2002.1592.View ArticleGoogle Scholar
- Didierlaurent L, Racine PJ, Houzet L, Chamontin C, Berkhout B, Mougel M: Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles. Nucleic Acids Res. 2011, 39: 8915-8927. 10.1093/nar/gkr577.PubMed CentralView ArticlePubMedGoogle Scholar
- Vrolijk MM, Harwig A, Berkhout B, Das AT: Destabilization of the TAR hairpin leads to extension of the polyA hairpin and inhibition of HIV-1 polyadenylation. Retrovirology. 2009, 6: 13-10.1186/1742-4690-6-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31: 3406-3415. 10.1093/nar/gkg595.PubMed CentralView ArticlePubMedGoogle Scholar
- Vrolijk MM, Ooms M, Harwig A, Das AT, Berkhout B: Destabilization of the TAR hairpin affects the structure and function of the HIV-1 leader RNA. Nucleic Acids Res. 2008, 36: 4352-4363. 10.1093/nar/gkn364.PubMed CentralView ArticlePubMedGoogle Scholar
- Huthoff H, Berkhout B: Two alternating structures for the HIV-1 leader RNA. RNA. 2001, 7: 143-157. 10.1017/S1355838201001881.PubMed CentralView ArticlePubMedGoogle Scholar
- Ooms M, Huthoff H, Russell R, Liang C, Berkhout B: A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions. J Virol. 2004, 78: 10814-10819. 10.1128/JVI.78.19.10814-10819.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Abbink TEM, Berkhout B: A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the gag start codon. J Biol Chem. 2003, 278: 11601-11611. 10.1074/jbc.M210291200.View ArticlePubMedGoogle Scholar
- Abbink TEM, Ooms M, Haasnoot PCJ, Berkhout B: The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation. Biochem. 2005, 44: 9058-9066. 10.1021/bi0502588.View ArticleGoogle Scholar
- Berkhout B, Ooms M, Beerens N, Huthoff H, Southern E, Verhoef K: In vitro evidence that the untranslated leader of the HIV-1 genome is an RNA checkpoint that regulates multiple functions through conformational changes. J Biol Chem. 2002, 277: 19967-19975. 10.1074/jbc.M200950200.View ArticlePubMedGoogle Scholar
- Lu K, Heng X, Garyu L, Monti S, Garcia EL, Kharytonchyk S, Dorjsuren B, Kulandaivel G, Jones S, Hiremath A, et al: NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging. Science. 2011, 334: 242-245. 10.1126/science.1210460.PubMed CentralView ArticlePubMedGoogle Scholar
- Andersen ES, Contera SA, Knudsen B, Damgaard CK, Besenbacher F, Kjems J: Role of the trans-activation response element in dimerization of HIV-1 RNA. J Biol Chem. 2004, 279: 22243-22249. 10.1074/jbc.M314326200.View ArticlePubMedGoogle Scholar
- Song R, Kafaie J, Laughrea M: Role of the 5′ TAR stem-loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA. Biochem. 2008, 47: 3283-3293. 10.1021/bi7023173.View ArticleGoogle Scholar
- Jalalirad M, Saadatmand J, Laughrea M: Dominant role of the 5’ TAR bulge in dimerization of HIV-1 genomic RNA, but no evidence of TAR-TAR kissing during in vivo virus assembly. Biochem. 2012, 51: 3744-3758. 10.1021/bi300111p.View ArticleGoogle Scholar
- Berkhout B: Multiple biological roles associated with the repeat (R) region of the HIV-1 RNA genome. Adv Pharmacol. 2000, 48: 29-73.View ArticlePubMedGoogle Scholar
- Das AT, Harwig A, Vrolijk MM, Berkhout B: The TAR hairpin of human immunodeficiency virus type-1 can be deleted when not required for Tat-mediated activation of transcription. J Virol. 2007, 81: 7742-7748. 10.1128/JVI.00392-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Berkhout B, Vastenhouw NL, Klasens BI, Huthoff H: Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription. RNA. 2001, 7: 1097-1114. 10.1017/S1355838201002035.PubMed CentralView ArticlePubMedGoogle Scholar
- Vo MN, Barany G, Rouzina I, Musier-Forsyth K: HIV-1 nucleocapsid protein switches the pathway of transactivation response element RNA/DNA annealing from loop-loop “kissing” to “zipper”. J Mol Biol. 2009, 386: 789-801. 10.1016/j.jmb.2008.12.070.PubMed CentralView ArticlePubMedGoogle Scholar
- Vo MN, Barany G, Rouzina I, Musier-Forsyth K: Mechanistic studies of mini-TAR RNA/DNA annealing in the absence and presence of HIV-1 nucleocapsid protein. J Mol Biol. 2006, 363: 244-261. 10.1016/j.jmb.2006.08.039.View ArticlePubMedGoogle Scholar
- Godet J, de RH, Raja C, Glasser N, Ficheux D, Darlix JL, Mely Y: During the early phase of HIV-1 DNA synthesis, nucleocapsid protein directs hybridization of the TAR complementary sequences via the ends of their double-stranded stem. J Mol Biol. 2006, 356: 1180-1192. 10.1016/j.jmb.2005.12.038.View ArticlePubMedGoogle Scholar
- Harrich D, Hooker CW, Parry E: The human immunodeficiency virus type 1 TAR RNA upper stem-loop plays distinct roles in reverse transcription and RNA packaging. J Virol. 2000, 74: 5639-5646. 10.1128/JVI.74.12.5639-5646.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Laughrea M, Jette L, Mak J, Kleiman L, Liang C, Wainberg MA: Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization. J Virol. 1997, 71: 3397-3406.PubMed CentralPubMedGoogle Scholar
- McBride MS, Panganiban AT: The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol. 1996, 70: 2963-2973.PubMed CentralPubMedGoogle Scholar
- Clever JL, Eckstein DA, Parslow TG: Genetic dissociation of the encapsidation and reverse transcription functions in the 5′R region of human immunodeficiency virus type 1. J Virol. 1999, 73: 101-109.PubMed CentralPubMedGoogle Scholar
- Houzet L, Paillart JC, Smagulova F, Maurel S, Morichaud Z, Marquet R, Mougel M: HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms. Nucleic Acids Res. 2007, 35: 2695-2704. 10.1093/nar/gkm153.PubMed CentralView ArticlePubMedGoogle Scholar
- Lever AM: HIV RNA packaging and lentivirus-based vectors. Adv Pharmacol. 2000, 48: 1-28.View ArticlePubMedGoogle Scholar
- Vogt PK: Historical Introduction to the general properties of retroviruses. In Retroviruses. Edited by: Coffin JM, Hughes SH, Varmus HE. 1997, Cold Spring Harbor Laboratory Press, New York, 1-25.Google Scholar
- McBride MS, Panganiban AT: Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo. J Virol. 1997, 71: 2050-2058.PubMed CentralPubMedGoogle Scholar
- Song R, Kafaie J, Yang L, Laughrea M: HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization. J Mol Biol. 2007, 371: 1084-1098. 10.1016/j.jmb.2007.06.010.View ArticlePubMedGoogle Scholar
- Ji S, Iwamoto A, Shioda T: Dissociation of genome dimerization from packaging functions and virion maturation of human immunodeficiency virus type 1. J Virol. 2002, 76: 959-967. 10.1128/JVI.76.3.959-967.2002.View ArticleGoogle Scholar
- Shen N, Jette L, Liang C, Wainberg MA, Laughrea M: Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging. J Virol. 2000, 74: 5729-5735. 10.1128/JVI.74.12.5729-5735.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Jalalirad M, Laughrea M: Formation of immature and mature genomic RNA dimers in wild-type and protease-inactive HIV-1: differential roles of the Gag polyprotein, nucleocapsid proteins NCp15, NCp9, NCp7, and the dimerization initiation site. Virol. 2010, 407: 225-236. 10.1016/j.virol.2010.08.013.View ArticleGoogle Scholar
- Berkhout B, van Wamel JLB: Role of the DIS hairpin in replication of human immunodeficiency virus type 1. J Virol. 1996, 70: 6723-6732.PubMed CentralPubMedGoogle Scholar
- Sakuragi JI, Ueda S, Iwamoto A, Shioda T: Possible role of dimerization in human immunodeficiency virus type 1 genome RNA packaging. J Virol. 2003, 77: 4060-4069. 10.1128/JVI.77.7.4060-4069.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Russell RS, Hu J, Beriault V, Mouland AJ, Laughrea M, Kleiman L, Wainberg MA, Liang C: Sequences downstream of the 5′ splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA. J Virol. 2003, 77: 84-96. 10.1128/JVI.77.1.84-96.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Moore MD, Fu W, Nikolaitchik O, Chen J, Ptak RG, Hu WS: Dimer initiation signal of human immunodeficiency virus type 1: its role in partner selection during RNA copackaging and its effects on recombination. J Virol. 2007, 81: 4002-4011. 10.1128/JVI.02589-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Moore MD, Nikolaitchik OA, Chen J, Hammarskjold ML, Rekosh D, Hu WS: Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses. PLoS Pathog. 2009, 5: e1000627-10.1371/journal.ppat.1000627.PubMed CentralView ArticlePubMedGoogle Scholar
- Russell RS, Liang C, Wainberg MA: Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?. Retrovirology. 2004, 1: 23-10.1186/1742-4690-1-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Paillart J-C, Berthoux L, Ottmann M, Darlix J-L, Marquet R, Ehresmann B, Ehresmann C: A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis. J Virol. 1996, 70: 8348-8354.PubMed CentralPubMedGoogle Scholar
- Das AT, Klaver B, Berkhout B: A hairpin structure in the R region of the human immunodeficiency virus type 1 RNA genome is instrumental in polyadenylation site selection. J Virol. 1999, 73: 81-91.PubMed CentralPubMedGoogle Scholar
- Hill MK, Shehu-Xhilaga M, Campbell SM, Poumbourios P, Crowe SM, Mak J: The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells. J Virol. 2003, 77: 8329-8335. 10.1128/JVI.77.15.8329-8335.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- StLouis DC, Gotte D, Sanders-Buell E, Ritchey DW, Salminen MO, Carr JK, McCutchan FE: Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection. J Virol. 1998, 72: 3991-3998.Google Scholar
- Centlivre M, Klaver B, Berkhout B, Das AT: Functional analysis of the complex trans-activating response element RNA structure in simian immunodeficiency virus. J Virol. 2008, 82: 9171-9178. 10.1128/JVI.00530-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Heng X, Kharytonchyk S, Garcia EL, Lu K, Divakaruni SS, Lacotti C, Edme K, Telesnitsky A, Summers MF: Identification of a minimal region of the HIV-1 5′-leader required for RNA dimerization, NC binding, and packaging. J Mol Biol. 2012, 417: 224-239. 10.1016/j.jmb.2012.01.033.PubMed CentralView ArticlePubMedGoogle Scholar
- Harrich D, Mavankal G, Mette-Snider A, Gaynor RB: Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication. J Virol. 1995, 69: 4906-4913.PubMed CentralPubMedGoogle Scholar
- Klaver B, Berkhout B: Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus. EMBO J. 1994, 13: 2650-2659.PubMed CentralPubMedGoogle Scholar
- Auersperg N: Long-term cultivation of hypodiploid human tumor cells. J Nat Cancer Inst. 1964, 32: 135-163.PubMedGoogle Scholar
- Das AT, Zhou X, Vink M, Klaver B, Verhoef K, Marzio G, Berkhout B: Viral evolution as a tool to improve the tetracycline-regulated gene expression system. J Biol Chem. 2004, 279: 18776-18782. 10.1074/jbc.M313895200.View ArticlePubMedGoogle Scholar
- Jeeninga RE, Jan B, Van den Berg H, Berkhout B: Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias. Retrovirology. 2006, 3: 64-10.1186/1742-4690-3-64.PubMed CentralView ArticlePubMedGoogle Scholar
- Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, Van der Noordaa J: Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990, 28: 495-503.PubMed CentralPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.