The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
© Pachulska-Wieczorek et al. 2016
Received: 16 October 2015
Accepted: 17 February 2016
Published: 17 March 2016
The Gag polyprotein is a multifunctional regulator of retroviral replication and major structural component of immature virions. The nucleic acid chaperone (NAC) activity is considered necessary to retroviral Gag functions, but so far, NAC activity has only been confirmed for HIV-1 and RSV Gag polyproteins. The nucleocapsid (NC) domain of Gag is proposed to be crucial for interactions with nucleic acids and NAC activity. The major function of matrix (MA) domain is targeting and binding of Gag to the plasma membrane but MA can also interact with RNA and influence NAC activity of Gag. Here, we characterize RNA binding properties and NAC activity of HIV-2 MA and Gag, lacking p6 domain (GagΔp6) and discuss potential contribution of NC and MA domains to HIV-2 GagΔp6 functions and interactions with RNA.
We found that HIV-2 GagΔp6 is a robust nucleic acid chaperone. HIV-2 MA protein promotes nucleic acids aggregation and tRNALys3 annealing in vitro. The NAC activity of HIV-2 NC is affected by salt which is in contrast to HIV-2 GagΔp6 and MA. At a physiological NaCl concentration the tRNALys3 annealing activity of HIV-2 GagΔp6 or MA is higher than HIV-2 NC. The HIV-2 NC and GagΔp6 show strong binding to the packaging signal (Ψ) of HIV-2 RNA and preference for the purine-rich sequences, while MA protein binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 GagΔp6 and NC promote HIV-2 RNA dimerization while our data do not support MA domain participation in this process in vitro.
We present that contrary to HIV-1 MA, HIV-2 MA displays NAC activity and we propose that MA domain may enhance the activity of HIV-2 GagΔp6. The role of the MA domain in the NAC activity of Gag may differ significantly between HIV-1 and HIV-2. The HIV-2 NC and MA interactions with RNA are not equivalent. Even though both NC and MA can facilitate tRNALys3 annealing, MA does not participate in RNA dimerization in vitro. Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag differs not only between, but also within, retroviral genera.
The retroviral Gag polyprotein is not only a major structural component of immature virions, but also acts as multifunctional regulator of virus replication [1–4]. The Gag polyproteins of human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) share 50 % sequence homology and each contains the N-terminal matrix (MA) domain, which is responsible for Gag targeting and binding to the plasma membrane [5, 6], the capsid domain (CA), which facilitates Gag multimerization [4, 7], and the nucleocapsid (NC) domain that interacts with viral and cellular nucleic acids (NA) [2, 3, 8]. Gag also contains two spacer regions (SP1, SP2) and the p6 domain located at the C-terminus, which is necessary for virus release from the infected cell . Before or shortly after virion release, the Gag polyprotein is cleaved in a highly ordered manner by viral protease into freestanding MA, CA, and NC proteins [1, 10]. In addition to the structural function in mature virions , the NC protein plays an important role in the facilitation of nucleic acid strand transfers during reverse transcription . Mature MA remains bound to the viral membrane , but it was proposed that a fraction of HIV-1 MA also enters newly infected cells, associates with the pre-integration complex (PIC), and affects proviral DNA circularization and integration [12, 13].
At a late stage in the HIV replication cycle, Gag may be responsible for the annealing of tRNALys3 to an 18-nt primer binding sequence (PBS) localized in the 5′UTR of the viral RNA, where tRNALys3 primes reverse transcription [2, 14]. The NAC activity is considered necessary to anneal tRNALys3 but, so far, it has only been confirmed for HIV-1 and Rous sarcoma virus (RSV) Gag proteins in vitro [15–18]. Chaperone proteins can facilitate folding and formation of the most thermodynamically favoured structures of nucleic acids . The NAC activity of HIV-1 Gag has been shown to depend on the NC domain, which is required for tRNALys3/viral RNA complex formation [14, 16, 17], whereas the MA domain can inhibit this process via RNA binding . Numerous lines of evidence support the nucleic-acid-binding properties of retroviral MA [15, 20–27], but significant differences in the role of MA domain in the overall NAC activity of Gag were observed between retroviral genera. In contrast to HIV-1, the chaperone activity of alpharetrovirus RSV Gag is independent of the MA domain . Moreover, HIV-1 and RSV NC display robust chaperone activity [15–17, 28], whereas MA proteins from both viruses do not promote annealing of primer tRNA in vitro [15–17]. The RNA-binding properties and chaperone activity of HIV-2 Gag have not been studied and the contribution of NC and MA domains remains undefined. We recently reported that HIV-2 NC is an effective NAC, but its activity is limited by the structural stability of the nucleic acid molecule to a much greater degree than that of HIV-1 NC .
As a nucleic acid chaperone, Gag binds NA non-specifically [2, 3], but is also engaged in highly specific recognition of cis-acting dimerization and packaging (Ψ) signals within the 5′UTR of the viral genomic RNA . Although HIV-1 Gag binds GU-rich sequences in the cytoplasm, its binding specificity changes to A-rich RNA motifs during virion assembly . The HIV-2 Gag-binding sites within the viral RNA remain uncharacterized and only limited information on HIV-2 NC binding to isolated domains of HIV-2 5′UTR in vitro is available [32, 33]. Similar packaging mechanisms are suggested for both viruses , but HIV-1 Gag is able to package HIV-2 RNA, whereas HIV-2 Gag cannot package HIV-1 RNA [35, 36]. The NC domains of HIV-1 and HIV-2 uncleaved Gag polyproteins are proposed to be crucial for the selection, dimerization, and packaging of viral RNA [8, 36–39]. In contrast, both NC and MA domains play direct roles in viral RNA packaging in deltaretroviruses [bovine leukaemia virus (BLV) and human T cell leukaemia virus type 2 (HTLV-2)] [22, 25, 26]. Interestingly, participation of the MA domain of HIV-1 Gag in these steps of viral replication was also suggested [23, 40, 41]. Moreover, an intriguing link between a mutation in the MA domain of HIV-2 Gag and viral RNA dimerization has been recently shown .
The three-dimensional structure of the entire HIV-1 and HIV-2 Gag is unknown, but the structures of their freestanding NC and MA have been presented [32, 43–46]. Moreover, the structure of the non-myristoylated HIV-1 Gag fragment (MA-CA-SP1-NC) was recently resolved by NMR spectroscopy . The mature NC proteins of HIV-1 and HIV-2 are small basic proteins, containing two zinc finger domains (ZFs). The ZFs are proposed to be crucial for specific interactions of NC with nucleic acids, whereas basic residues from the disordered N-terminus play a role in non-specific interactions and NAC activity [8, 11, 29, 47]. Despite the limited sequence similarity between HIV-1 MA and HIV-2 MA, both proteins are composed of six α-helices and three β-sheet elements, and are myristoylated at the N-terminus [43, 44, 46]. The myristyl group and amino acid residues of HIV-1 and HIV-2 MA are engaged in PM binding [5, 43]. Importantly, some of those residues are located within the highly basic region (HBR) at the N-terminus of MA, which is proposed to be important for interactions with RNA in HIV-1 [21, 48–50]. RNA binding to the MA domain ensures the specificity of HIV-1 Gag interactions with PM phospholipids [6, 21, 48, 51]. Whether the MA domain of HIV-2 Gag is involved in RNA binding is not known.
Within this work we characterized the RNA-binding properties and nucleic acid chaperone activity of recombinant HIV-2 GagΔp6, NC and MA proteins. We identified binding sites of HIV-2 GagΔp6 and isolated NC and MA domains within the 5′UTR of HIV-2 RNA. Both HIV-2 NC and GagΔp6 show strong binding to the packaging signal and preference for the purine-rich sequences, while HIV-2 MA binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 NC promotes HIV-2 RNA dimerization while this process is not supported by HIV-2 MA, suggesting that MA domain is dispensable for HIV-2 GagΔp6-promoted dimerization in vitro. We found that HIV-2 GagΔp6 is a robust nucleic acid chaperone and we propose that both NC and MA domains contribute to nucleic acids aggregation and tRNALys3 annealing in vitro. The NAC activity of HIV-2 NC is affected by salt in contrast to that of HIV-2 GagΔp6 and MA.
Nucleic acid-binding properties of HIV-2 GagΔp6, MA, and NC
Calculated dissociation constants for HIV-2 NC, MA, and GagΔp6 complexes with selected RNA
207 ± 9
431 ± 88
49 ± 5
285 ± 21
417 ± 74
52 ± 5
385 ± 16
430 ± 46
53 ± 5
541 ± 6
538 ± 68
65 ± 4
679 ± 16
593 ± 76
97 ± 7
1432 ± 22
2377 ± 209
965 ± 83
36 ± 3
335 ± 28
31 ± 1
117 ± 6
289 ± 4
31 ± 3
196 ± 9
290 ± 5
30 ± 2
277 ± 26
340 ± 25
31 ± 2
392 ± 37
382 ± 25
41 ± 3
2036 ± 212
2072 ± 98
314 ± 29
7 ± 2
413 ± 52
27 ± 1
25 ± 1
300 ± 41
17 ± 1
71 ± 1
312 ± 29
15 ± 1
135 ± 4
357 ± 21
24 ± 2
240 ± 10
374 ± 19
26 ± 3
979 ± 26
1514 ± 210
125 ± 5
HIV-2 MA displays high TAR annealing activity
Annealing parameters of HIV-2 NC, MA, GagΔp6
HIV-2 protein (μM)
NA annealed (%)
TAR annealing (20 mM NaCl)
2.0 ± 0.40
95.3 ± 0.7
0.6 ± 0.05
91.1 ± 2.4
0.9 ± 0.11
92.5 ± 1.3
tRNALys3 annealing (20 mM NaCl)
1.87 ± 0.44
95.1 ± 1.8
0.17 ± 0.08
37.8 ± 5.0
0.18 ± 0.06
85.1 ± 5.2
tRNALys3 annealing (150 mM NaCl)
0.07 ± 0.03
35.8 ± 4.9
0.21 ± 0.06
48.1 ± 4.9
0.26 ± 0.03
82.1 ± 3.4
0.11 ± 0.02
43 ± 5.2
0.22 ± 0.07
60 ± 4.9
0.37 ± 0.07
90 ± 3.7
RNA dimerization (150 mM NaCl)
0.48 ± 0.10
49.2 ± 1.6
0.15 ± 0.01
45.7 ± 2.2
HIV-2 MA effectively aggregates nucleic acids
The ability to sequence non-specific aggregation of NA is considered an important characteristic of NAC proteins . We directly compared the NA-aggregation properties of analysed HIV-2 proteins and HIV-1 MA using sedimentation assays (Fig. 3c). In this assay, the 32P-labelled HIV-1 TAR(−) DNA and TAR RNA were incubated with increasing concentrations of protein, centrifuged, and the amount of non-aggregated NA was measured. We found that HIV-2 NC, MA, and GagΔp6 are effective NA-aggregating agents, since ~80 % of NA aggregation was detected at a 0.2 µM concentration of each protein. The observed NA aggregation at a given protein concentration was similar for HIV-2 NC and MA, but significantly greater for HIV-2 GagΔp6, since a two-fold lower concentration of GagΔp6 was sufficient for the maximal NA aggregation. HIV-1 MA aggregated NA much weaker than HIV-2 proteins since only up to ~40 % of aggregation was detected at the highest protein concentration used (0.8 µM).
NC and MA domains may contribute to the tRNALys3 annealing activity of HIV-2 Gag
The HIV-1 Gag and NC, via their NAC activity, mediate tRNALys3 annealing in vitro and in vivo [14, 17, 36]. On the contrary, HIV-1 MA does not promote the tRNALys3 annealing in vitro even when high MA concentrations and long reaction times were employed . The aggregation and TAR annealing assays demonstrated that HIV-2 MA displays high NAC activity in vitro (Fig. 3). To further characterize and compare the activity of the analysed HIV-2 proteins, gel-shift tRNALys3 annealing assays were performed, using the in vitro transcribed, unmodified tRNALys3, and a 560-nt RNA, corresponding to the 5′UTR of HIV-2 genomic RNA (Fig. 1b). To determine the influence of salt on the NAC activity of proteins, the annealing reactions were conducted at low (20 or 50 mM) and physiological (150 mM) NaCl concentrations.
The time course tRNALys3 annealing assays performed at 1.5 µM protein concentration (1 protein per 3.9 nt), revealed important differences between the annealing rates of HIV-2 NC, MA, and GagΔp6 at 20 mM and 150 mM NaCl (Fig. 4c, d). At low NaCl, HIV-2 NC displayed a significantly higher (~11-fold) annealing rate than HIV-2 GagΔp6 or MA. The observed final percentages of tRNALys3 annealed were similar for HIV-2 NC and GagΔp6 (~95 and ~85 %, respectively), and ~38 % in the presence of HIV-2 MA. At physiological NaCl concentration, the annealing rates and final percentages of annealing in the presence of HIV-2 MA and GagΔp6 did not change significantly. Interestingly, HIV-2 NC exhibited an almost 27-fold lower annealing rate and the final percentage of tRNALys3 annealed was reduced to ~38 %. The increase in HIV-2 proteins concentration to 3 µM (1 protein per 1.8 nt) did not change the observed trend and inhibitory effect of 150 mM NaCl was still observed for HIV-2 NC but not for HIV-2 MA or GagΔp6 (Table 2).
HIV-2 MA does not promote HIV-2 RNA dimerization
Discussion and conclusions
In this work, we investigated the chaperone activity of HIV-2 GagΔp6, MA, and NC proteins, their binding specificity, and interactions with HIV-2 RNA. We also included HIV-1 MA in chaperone assays for direct comparison. The results of NA aggregation, TAR, or tRNALys3 annealing assays showed that, on a molar basis, HIV-2 GagΔp6 is a more robust nucleic acid chaperone than NC. Moreover, at a physiological salt concentration, the rate and final percentage of annealed tRNALys3 were significantly higher in the presence of HIV-2 GagΔp6 than HIV-2 NC (Fig. 4b, c; Table 2). The salt-dependent binding assays revealed that HIV-2 GagΔp6 binds to RNA with higher affinity than freestanding HIV-2 NC (Table 1). These observations suggest that domains other than NC contribute to the NAC activity of HIV-2 Gag. Indeed, we found that HIV-2 MA binds RNA and displays high NAC activity in vitro, since it effectively aggregated NA and facilitated the annealing of TAR hairpins (Fig. 3). This is in contrast to HIV-1 MA, which displays very poor NA aggregation and TAR annealing activity (Fig. 3) . In addition HIV-1 MA does not chaperone tRNALys3 annealing in vitro while in the presence of HIV-2 MA, up to ~50 % of tRNALys3 annealing was measured (Fig. 4). In low salt concentration HIV-2 MA displays reduced tRNALys3 annealing activity compared to that of HIV-2 NC and GagΔp6. However the difference in activity is less evident in TAR annealing assays, suggesting that NAC activity of HIV-2 MA is limited by substrates length and stability to a greater degree than that of HIV-2 NC or GagΔp6.
We observed that HIV-2 GagΔp6 and MA binding to RNA is salt-independent in the range from 50 to 250 mM NaCl (Table 1). Consistently with these results, the tRNALys3 annealing activity of HIV-2 GagΔp6 and MA was not sensitive to monovalent salt at 20–150 mM (Fig. 4). Contrary to our data on HIV-2 MA, the increase in monovalent salt concentration from 50 to 150 mM significantly decreased the RNA-binding affinity of HIV-1 MA . The RNA-binding properties and NAC activity of HIV-2 NC are highly salt-sensitive. At a physiological NaCl concentration, the extent and rate of tRNALys3 annealing in the presence of HIV-2 NC were lower than in the presence of HIV-2 MA (Fig. 4; Table 2). The comparison of the HIV-2 NC and MA chaperone activity at different salt concentrations supports involvement of both RNA-binding domains of HIV-2 GagΔp6 in tRNALys3 annealing.
Based on the presented results, we propose that both NC and MA domains contribute to the chaperone activity of HIV-2 Gag. Although HIV-2 NC is an effective chaperone, its activity is lower than that of HIV-1 NC . Additionally to the NC domain, the MA domain via interactions with RNA and its NAC activity may enhance the activity of HIV-2 Gag. However we cannot exclude influence of other domains or multimerization on the NAC activity of HIV-2 Gag. The available data indicate that for HIV-1 the NC domain is primarily responsible for the overall NAC activity of HIV-1 Gag [16, 17], whereas the MA domain via RNA binding inhibits the NAC activity of HIV-1 Gag . Interestingly, the MA domain does not influence the NAC activity of RSV Gag (alpharetrovirus) , while the HTLV-2 MA protein (deltaretrovirus) displays significantly higher chaperone activity than HTLV-2 NC . Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag may differ not only between, but also within, retroviral genera.
A recent study has shown that HIV-1 Gag exhibits significant differences in salt-dependent binding to diverse HIV-1 RNA fragments and binds non-Ψ RNA with low specificity via its NC and MA domains, whereas binding to Ψ RNA is highly specific and only the NC domain is engaged . Such a binding model is not common to all retroviral Gag polyproteins, since, in deltaretroviruses (HTLV-2), the MA domain binds RNA more specifically than NC and plays a dominant role in the initial recognition of the Ψ signal in genomic RNA . Our results demonstrate that HIV-2 GagΔp6 binds both Ψ and non-Ψ RNAs with high specificity, which was manifested by a negligible change in the dissociation constants within the 50–250 mM NaCl range (Table 1). Moreover, even at 500 mM NaCl, HIV-2 GagΔp6 interacted with Ψ RNA and non-Ψ (PBS) RNA with strong affinities (Kd ≈ 125 and 314 nM, respectively). HIV-2 GagΔp6 binding to Ψ RNA was similar to that presented for HIV-1 GagΔp6, but HIV-1 GagΔp6 did not bind non-Ψ RNA (TARpA) at 500 mM NaCl . HIV-2 GagΔp6 bound TARpA at 500 mM NaCl but with affinity (Kd ≈ 965 nM) lower than PBS. At low ionic strength (50 mM NaCl), HIV-2 NC binding to Ψ RNA was comparable to that of HIV-2 GagΔp6, but highly susceptible to salt concentration. HIV-2 NC and MA bound non-Ψ RNA with comparable affinity, but MA binding to Ψ RNA was notably weaker than that of NC or GagΔp6. Taken together, our results suggest contributions of both the NC and MA domains to the interactions of HIV-2 Gag with RNA, but the NC domain plays a major role in recognizing the Ψ signal. Our results showing that HIV-2 GagΔp6 and NC, but not MA, occupy some of the sites within the Ψ region of 5′UTR may further support this notion. Although the majority of HIV-2 GagΔp6, NC, and MA binding sites cluster within the Ψ region of HIV-2 5′UTR, extensive interactions were also detected within the TAR and PBS domains (Fig. 2). A recent study demonstrated that the RNA-binding specificity of HIV-1 Gag changes during viral replication . Only a few regions of the viral RNA interacted with HIV-1 Gag in cytosol, including the 5′UTR, while the entire RNA was covered within the virus particles. Interestingly, the binding of HIV-1 Gag to TAR observed in the cytosol was HIV-1 subtype-dependent. Several lines of evidence suggested that TAR might be important for HIV dimerization and packaging [61, 62]. Moreover, TAR stability is considered important in the strand transfer during reverse transcription  and may also influence the Gag translation efficiency . Our in vitro conditions are likely in favour of the detection of the high-affinity binding sites, but without differentiation of the replication stage. Binding of HIV-2 GagΔp6, NC, and MA to PBS in the vicinity of the tRNALys3 binding regions may support their involvement in primer annealing. Indeed, all proteins promoted tRNALys3 annealing to the 5′UTR (Fig. 4). Interestingly, findings that HIV-1 Gag has a strong preference for G-rich binding sites in cells and A-rich in virions  is reflected in our in vitro binding studies, showing a high purine content within the HIV-2 GagΔp6 binding sites.
In HIV-2 RNA, packaging and dimerization signals overlap, and the NC domain of HIV-2 Gag is proposed to be required for viral genome dimerization and packaging in vivo [37, 38, 56]. Indeed, we found that HIV-2 NC and GagΔp6 effectively promote in vitro dimerization of HIV-2 RNA containing DIS and pal dimerization signals (Fig. 5). For HIV-1 Gag, numerous lines of evidence support the involvement of the NC domain in the genomic RNA selection, dimerization, and packaging, but several observations suggest a contribution of the MA domain to these processes . It was found that the presence of either the NC or the MA domain is required for genome packaging during HIV-1 particle assembly [23, 40]. However, a recent study revealed that the MA domain of HIV-1 Gag binds almost exclusively to specific cellular tRNAs . Our data suggest that the MA domain is dispensable for HIV-2 GagΔp6-promoted dimerization, since this process is not supported by HIV-2 MA in vitro. On the other hand, HIV-2 MA binds some cis-acting dimerization and packaging sequences in 5′UTR of HIV-2 RNA (Fig. 2). Therefore, we cannot exclude the participation of the MA domain in HIV-2 genome selection and dimerization in the cell.
Cloning, expression and purification of recombinant proteins
The HIV-2ROD NC protein was obtained using pGEX-4T-3-NCp8 as described previously . Sequences encoding HIV-2 MA, GagΔp6 (HIV-2ROD isolate) and HIV-1 MA (HIV-1NL4-3 isolate) were PCR amplified from HIV-2 pROD10-EVA232 and pNL4-3-ARP2006 (National Institute for Biological Standards and Control, Centre for AIDS Reagents, UK). PCR products were digested, purified using PureLink® spin columns (Invitrogen), and cloned into a pGEX-4T-3 expression vector. The sequence of each construct was confirmed by DNA sequencing. The glutathione S-transferase (GST) fusion HIV-2 NC, MA, and GagΔp6 and HIV-1 MA recombinant proteins were expressed in One Shot® BL21(DE3)pLysS E. coli (Invitrogen) and purified by affinity chromatography on Glutathione Sepharose (GE Healthcare) as described in the Additional file 5. The GST tag was removed by thrombin cleavage. The purity of proteins was assessed by SDS–PAGE and estimated to be above 90 %. Protein concentrations were determined by their absorption spectrum and protein samples were aliquoted and stored at −80 °C.
DNA and RNA substrates
TAR(−) DNA, corresponding to the trans activation response (TAR) sequences of HIV-1MAL, was 32P-labelled at the 5′-end with [γ-32P]ATP using T4 polynucleotide kinase (Fermentas) and purified using NucAway Spin Columns (Life Technologies). HIV-1 TAR RNA and unmodified human tRNALys3 (referred to here as tRNALys3) were obtained using a PCR-generated template (Additional file 6) and Ambion T7-MEGAshortscript. Transcripts were purified by denaturing gel electrophoresis (8 M urea) in 1 × TBE, followed by elution and ethanol precipitation. The tRNALys3 was 3′-end labelled using [α-32P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). Templates for in vitro transcription of HIV-2 RNA molecules were obtained by PCR amplification of fragments from the HIV-2 plasmid pROD10-EVA232 using a forward primer containing a T7 promoter sequence (Additional file 6). The RNA molecules were as follows: TARpA (nt +1–188), PBS (nt +197–379), Ψ (nt +380–560), RNA +1–444, 5′UTR (nt +1–560) and RNA +1–891. RNAs were synthesized using T7-MEGAscript (Ambion) and purified using Direct-zol™ RNA MiniPrep (Zymo Research). The integrity of the RNAs was assessed by agarose gel electrophoresis under denaturing conditions. Purified RNA was stored at −20 °C. For some assays, RNA was 3′-end labelled using [α-32P]pCp and T4 RNA ligase (Fermentas) following purification using Direct-zol™ RNA MiniPrep (Zymo Research).
Equilibrium-binding experiments were performed as described previously  with the following modifications. Reactions were carried out in binding buffer (20 mM HEPES–KOH pH 7.5, 1 mM MgCl2, 10 µM TCEP, 5 mM β-mercaptoethanol, 10 µM ZnCl2, and 50–500 mM NaCl). The final concentration of RNA was 0.2 nM. The binding reactions were incubated for 25 min at room temperature and then 50 µl of each reaction was filtered and washed with 200 µl of binding buffer containing 50 mM NaCl. After filtration, the membranes were dried and exposed to a phosphoimager screen. Data were analysed using Multigauge (Fuji) and Origin (OriginLab) software.
Hydroxyl radical footprinting and detection of RNA cleavage products
RNA +1–891 was used for footprinting experiments (Additional file 6) and the secondary structure of the 5′UTR within this RNA was confirmed previously . RNA samples (5 pmol) were heated at 95 °C for 1 min and slowly cooled to 4 °C. Subsequently, buffer was added to the final concentration of 40 mM Tris–HCl, pH 8.0, 130 mM KCl, 0.5 mM EDTA, and 5 mM MgCl2, and samples were incubated for 25 min at 37 °C. Folded RNA samples were diluted 20-fold with 20 mM Tris–HCl, pH 8.0, followed by addition of NC, MA, or GagΔp6 (6 μl of 3 µM, 6 µM, or 12 µM protein in the buffer containing 50 mM Tris–HCl, pH 8.0, 1 M NaCl, 6.7 mM β-mercaptoethanol, 2.5 mM DTT, 0.1 mM ZnCl2) to a 70 μl reaction. RNA/protein complexes were formed at 0 °C for 20 min. Footprinting reactions were initiated by applying on the wall of the tube 1 μl of 2.5 mM (NH4)Fe(SO4)2, 50 mM sodium ascorbate, 1.5 % H2O2, and 2.75 mM EDTA, and centrifugating. After 15 s at 24 °C, reactions were quenched by the addition of 20 μl of stop solution containing 0.1 M thiourea and 0.2 M EDTA. RNA were purified using Direct-zol RNA MiniPrep Kit (Zymo Research). For the reverse transcription reactions, a total of 1.5 pmols of RNA was mixed with 2 µl of fluorescently labelled primer 186, 540, or 787 (Additional file 6) [4 μM Cy5 (with reagent) and 6 uM Cy5.5 (without reagent)] and 12 μl of primer-template solutions were incubated at 85 °C for 3 min, 60 °C for 5 min, 35 °C for 5 min, and 50 °C for 2 min. Reverse transcription and sample processing were carried out as previously described . Sequencing ladders were prepared using a Thermo Sequenase Cycle Sequencing Kit (Affymetrix) according to the manufacturer’s protocol. Samples and sequencing ladders were purified using a ZR DNA Sequencing Clean-up Kit (Zymo Research) and analysed on a GenomeLab GeXP Analysis System (Beckman-Coulter). Three to nine repetitions were obtained for each read. Electropherogram peaks were converted to reactivity values using Shapefinder software . Reverse transcription stops in the control reaction were identified as outlying high peaks in the plotted background area. To normalize the data, peak intensity for each nucleotide was divided by the average intensity of the 8 % most reactive peaks excluding outliers. The outliers were defined as greater than 1.5 times the interquartile difference above the 3rd quartile . Normalized data were averaged and nucleotide positions corresponding to reverse transcription stops were excluded from further analysis. Differences between reactivity values for reaction without protein and containing protein were calculated. The consistent drop in reactivity with increasing protein concentration larger than at least 20 % of reactivity value was regarded as a possible binding site.
To estimate number of binding sites within different domains of 5′UTR for HIV-2 GagΔp6, NC and MA, number of residues protected from hydroxyl radical cleavage in the presence of only GagΔp6 and NC were compared to the number of those protected only in the presence of GagΔp6 and MA.
TAR annealing assay
32P-labelled HIV-1 TAR(−) DNA (1 nM) and unlabelled HIV-1 TAR RNA (6 nM) were heat denaturated and folded separately in buffer containing 20 mM Tris–HCl, pH 7.5, 30 mM NaCl, 0.1 mM MgCl2, 10 µM ZnCl2, and 5 mM DTT. Then, the mixture of both oligonucleotides was incubated with increasing concentrations of each protein (0–0.8 µM) at 37 °C for 5 min. The time course assays were conducted at 37 °C using 0.2 µM protein and the samples were removed at the indicated time points. All reactions were quenched with 0.5 volume of stop solution (20 % glycerol, 20 mM EDTA pH 8.0, 0.1 % SDS, 0.25 % bromophenol blue, and 0.4 mg/ml yeast tRNA). Samples were analysed by native PAGE (8 %) in 0.5 × TBE at 4 °C.
32P-labelled HIV-1 TAR(−) DNA (1 nM) was combined with complementary unlabelled TAR RNA in a buffer containing 50 mM Tris pH 7.5, 20 mM NaCl, and 0.2 mM MgCl2. Reactions (10 µl) were incubated with increasing protein concentrations (0–0.8 µM) at 37 °C for 5 min. Subsequently, the mixtures were centrifuged at 11,400 rpm for 20 min. Supernatants (2 µl) were collected and subjected to scintillation counting.
tRNALys3 annealing assay
32P-labelled tRNALys3 (2 nM) and unlabelled +1–560 HIV-2 RNA (10 nM) were refolded in 50 mM Tris–HCl, pH 7.5 by heating at 95 °C for 1 min and slow cooling to 37 °C, followed by addition of MgCl2 to 10 mM and placement on ice. The annealing buffer contained 50 mM Tris–HCl pH 7.5, 5 mM DTT, and 1 mM MgCl2, and different NaCl concentrations of 20, 50, or 150 mM. The mixture was incubated at 37 °C for 10 min, followed by addition of protein and further incubation for 10 min. The time course assays were conducted at 37 °C using 1.5 or 3 µM protein and the aliquots were removed at the indicated time points. All reactions were quenched by incubation with 1 % (w/v) SDS at room temperature for 5 min. The samples were phenol/chloroform-extracted, mixed with loading buffer (50 % glycerol with dyes), and separated on 1.4 % SDS-agarose gel in 1 × TBE at room temperature.
The unlabelled +1–444 HIV-2 (400 nM) spiked with the trace amount of the same 32P-labelled transcript was heat denatured in 50 mM Tris–HCl pH 7.5, 40 mM KCl, 150 mM NaCl, and 0.1 mM MgCl2. The mixture was slowly cooled to 37 °C and placed on ice, followed by addition of different concentrations of proteins. The dimerization was allowed to proceed at 37 °C for 30 min. The time course dimerization assays were conducted at 37 °C using 6 µM protein and the aliquots were removed at the indicated time points. All dimerization reactions were quenched by incubation with 1 % (w/v) SDS at room temperature for 5 min, phenol/chloroform-extracted, and mixed with loading buffer (50 % glycerol with dyes). The products were separated on 1 % agarose gel in 1 × TBE at room temperature.
All gels were autoradiographed and quantitatively analysed by phosphorimaging using a FLA-5100 phosphorimager with MultiGaugeV 3.0 software (FujiFilm). The obtained data were analysed using Origin (OriginLab) software. All graphs represent averaged data from three or more independent experiments with standard deviations indicated. In all cases, at least three independent experiments were performed, and the data presented are representative of the whole.
KPW designed the study. KPW and KJP performed the experiments, analysed and interpreted the data and wrote the manuscript. LB and MB performed the experiments, analysed the data and helped to draft the manuscript. RWA provided important suggestions to the manuscript and helped to secure funding. All authors read and approved the final manuscript.
This work was supported by National Science Centre Poland [2011/01/D/NZ1/03478 to KPW, 2012/06/A/ST6/00384 to RWA] and Ministry of Science and Higher Education Poland [0397/IP1/2011/71 to KPW, 0492/IP1/2013/72 to KJP], and European Union Regional Development Fund and the Polish Ministry of Science and Higher Education, under the Leading National Research Centre (KNOW) Program. Funding for open access charge National Science Centre Poland [2012/06/A/ST6/00384].
The authors declare that they have no competing interests.
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