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Thermostable properties of the equine infectious anemia virus nucleocapsid protein NCp11
Biochemical and Biophysical Research Communications 510 (2019) 472e478
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Thermostable properties of the equine infectious anemia virus nucleocapsid protein NCp11 Jinzhong Wang a, b, c, Qinghua Wang a, Shasha Hao a, Chao Guo a, Jing An a, Qingmiao Zhang a, Ruonan Liang a, Ying Wang a, b, c, * a b c
TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, 300457, China Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, 23 Hongda Street, TEDA, Tianjin, 300457, China Tianjin Key Laboratory of Microbial Functional Genomics, 23 Hongda Street, TEDA, Tianjin, 300457, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 January 2019 Accepted 31 January 2019 Available online 5 February 2019
Retroviral nucleocapsid (NC) proteins are multifunctional nucleic acid binding proteins, playing critical roles in essentially every step of the viral replication cycle. As a small, basic protein, NC contains one or two highly conserved zinc-finger domains, each having an invariant CCHC motif, flanked by basic residues. In this study, we report for the first time, to our knowledge, the thermostable property of equine infectious anemia virus (EIAV) NCp11. About 43% of purified NCp11 remained soluble after incubation at 100 C for 60 min, and heat-treated NCp11 maintained its abilities to bind to the E. coli RNA and the EIAV packaging signal sequence. At a very high degree of sequence occupancy, NCp11 inhibited first-strand cDNA synthesis catalyzed by either a commercial or the purified EIAV reverse transcriptase, and heattreated NCp11 still inhibited the first-strand cDNA synthesis. We also found that protein concentrations, at a range from 0.1 to 0.9 mg/ml, have not affected the NCp11 thermostability significantly. However, NCp11 at acidic pH was more thermostable. Our findings highlight a new feature of the NC protein. Detailed understanding of NC's properties and functions will facilitate the development of effective and rational therapeutic strategies against retroviruses. © 2019 Elsevier Inc. All rights reserved.
Keywords: EIAV Nucleocapsid NCp11 Thermostable RNA binding First-strand cDNA synthesis
1. Introduction Retroviral nucleocapsid (NC) proteins are small, highly basic proteins with one or two zinc finger domains, each containing the invariant CCHC Zn2þ ion binding motif, flanked by basic residues [1,2]. The mature NC protein is derived from the Gag polyprotein which is composed of matrix, capsid, NC domains and a C-terminal peptide [3]. The NC domain is responsible for the recognition of viral packaging signal (psi, j), resulting in the specific encapsidation of two copies of genomic RNA (gRNA) into the viral particle [4]. Some mutations affecting the NC domain of human immunodeficiency virus (HIV) Gag reduce the virus infectivity dramatically [5]. During virion maturation, Gag polyproteins are cleaved by virus-encoded protease in a highly-regulated and well-ordered manner to generate viral structure proteins [6]. Among them, about 1500e2000 NC molecules are associated with the viral RNA,
* Corresponding author. TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, 300457, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.bbrc.2019.01.137 0006-291X/© 2019 Elsevier Inc. All rights reserved.
coating and protecting the entire dimeric gRNA in a histone-like manner [7]. NC protein can bind to different nucleic acid (NA) sequences at varying affinities [8], and is regarded as an ATPindependent NA chaperone [9,10]. At low degree of sequence occupancy when the NC:NA ratio is about 1:100 nt, NC binds specific viral sequences with high affinity; At high degree when the NC:NA ratio is from 1:15 to 1:7, NC binds NAs, causing their aggregation and driving the remodeling of the NC-NA complexes to reach compact and stable structures; At very high degree when NC:NA ratio is from 1:5 to 1:1, NC coats NAs extensively, providing protection to, or even freezes the compact nucleoprotein complexes [7]. Upon entry into the host cell, the NA chaperone activity of HIV NC is critical for its regulation of the reverse transcription. NC stabilizes interactions between tRNA primer and gRNA template, increases the efficiency of minus-strand transfer, helps the reverse transcriptase to destabilize RNA secondary structure, and contributes to the overall plus-strand transfer [9]. Besides, HIV NC also stimulates the integration of cDNA in vitro [11].
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As a multifunctional nucleic acid binding protein playing critical roles in essentially every step of the viral replication cycle, NC has been regarded as an ideal target for antiviral therapy [12], and has attracted more and more attention. Equine infectious anaemia virus (EIAV) was the first-reported lentivirus and retrovirus [13]. Compared with other lentiviruses, EIAV has the smallest genome size, encodes fewer regulatory proteins, and the first successful lentiviral vaccine was developed for EIAV [13,14]. In this study, the EIAV NCp11 was investigated, and for the first time, to our knowledge, the thermostability of NCp11 was found. Heat-treated NCp11 maintained its abilities to bind to E. coli RNA or EIAVpsi, maintained its ability to inhibit the first-strand cDNA synthesis catalyzed by either a commercial or the EIAV reverse transcriptase. Detailed understanding of NC's properties will benefit the understanding of its functions in viral replication and facilitate the development of effective and rational therapeutic strategies against retroviruses. 2. Materials and methods 2.1. Plasmids Plasmids pET28-NCp11 and pET28-p9 were described previously [15]. Plasmid pBS-EIAVpsi was constructed, which contains the EIAVuk genome (AF016316.1) from nt 287 to nt 546 covering the EIAVpsi sequence [16,17], using primers listed in the Supplementary. Coding regions of the EIAV reverse transcriptase p66 and p51 were amplified and cloned into pET32 and pET28 vectors, respectively. All plasmids constructed in this study were confirmed by DNA sequencing. 2.2. Protein purification, heat treatment and SDS-PAGE E. coli BL21 (DE3) harboring pET28-NCp11 or pET28-p9 was
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induced with 0.1 mM isopropylthio-b-D-galactoside (IPTG) for 4 h at 37 C, and that harboring pET32-EIAVp66 or pET28EIAVp51 was induced with 0.05 mM IPTG for 6 h at room temperature. 6 histidine-tagged proteins were purified as described [15]. In some experiments, before or after the cell lysate was employed onto the HIS-Select® Nickel Affinity Gel (Sigma-Aldrich), 20 mg/ml RNase A was added and samples were incubated on ice or at 37 C for 30e60 min. Purified NCp11, p9 or the equimolar mixture of p66 and p51 were dialyzed overnight against phosphate-buffered saline (PBS) unless otherwise specified. Protein concentrations were determined by bicinchoninic acid assay (Thermo). To determine the thermostability of NCp11, purified NCp11 or sonicated clear cell lysate of IPTG-induced E. coli BL21 (DE3) was incubated at 100 C for 0, 5, 10, 30 or 60 min, respectively, and then centrifuged to remove precipitated proteins. Purified NCp11 at different concentrations (0.1, 0.3 or 0.9 mg/ml), and NCp11 dialyzed against 20 mM acetate buffer (pH 5 or 4), phosphate buffer (pH 8, 7 or 6) [18] or PBS supplemented with 10 mM EDTA were also heated. SDS-PAGE was performed as described [15]. The levels of soluble NCp11 retained were quantified using the ImageJ software and normalized to the NCp11 level without heating [19]. 2.3. The far UV circular dichroism (CD) spectroscopic analysis Purified NCp11 was heated, clarified, adjusted to 0.2 mg/ml with PBS. Protein samples were transferred into a quartz glass cuvette with a 1 mm path length to a Jasco J-715 spectropolarimeter (Jasco, Japan) which was purged continuously with nitrogen, and scanned at 50 nm/min at 25 C over a wavelength range of 190e260 nm. Results of the CD measurements were converted to the mean residue ellipticities q [20].
Fig. 1. NCp11 is thermostable. A. E. coli BL21 (DE3) transformed with pET28-NCp11 was uninduced (lane 2) or induced with 0.1 mM IPTG for 4 h (lanes 3e8). Total (lanes 2e3) and soluble proteins heated at 100 C for 0 (lane 4), 5 (lane 5), 10 (lane 6), 30 (lane 7) or 60 min (lane 8) were analyzed by 12% SDS-PAGE. B. Purified NCp11 was incubated at 100 C for 0 (lane 2), 5 (lane 3), 10 (lane 4), 30 (lane 5) or 60 min (lane 6) and analyzed by SDS-PAGE. C. Quantitative results of B with three repeats. D. Circular dichroism spectra of NCp11 with or without heat treatment. E. Purified NCp11 was incubated at 0 C, 37 C or 60 C, respectively, and analyzed by SDS-PAGE. F. Quantitative measurements of E.
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2.4. In vitroRNA binding and electrophoretic mobility shift assay (EMSA) Total RNA was extracted from E. coli BL21 (DE3) using the Ultrapure RNA Kit (CoWin Biosciences), and quantified with NanoDrop ND-1000. For in vitro NCp11-RNA binding experiments, binding buffer (10 mM Hepes, pH 7.9, 50 mM potassium chloride, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM magnesium chloride and 0.5 mM PMSF) was used, and reaction mixtures were incubated at room temperature for 10 min. Samples were analyzed by EMSA [21] using 0.7% agarose gel in 1 TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) containing 0.1 mg/ml ethidium bromide. Images were acquired with the UV transilluminator, and shifted bands were quantified with the ImageJ software. 2.5. PCR and RT-PCR E. coli BL21 (DE3) was transformed with pET28-NCp11 or cotransformed with pET28-NCp11 and pBS-EIAVpsi. NCp11-RNA complexes were purified by nickel affinity chromatography, and RNA in the complexes was extracted. RT-PCR or PCR was performed using the SuperRT One Step RT-PCR Kit or the 2 Es Taq Master Mix (CoWin Biosciences), amplifying a 124 bp fragment (nt 338 to nt
461 of EIAVuk). Primers are listed in the Supplementary. For some experiments, 0.5 U Recombinant DNase I (RNase-free) and 5 U RNase Inhibitor (TaKaRa) were utilized to remove the first-strand cDNA. If the EIAV reverse transcriptase was used, 10 ml reaction mixture containing 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.2 mM dNTPs, 20 U RNase Inhibitor, 1 mM of each primer, 50 ng purified p66/p51 heterodimer and 40 ng template RNA was incubated at 37 C for 30 min. After first-strand cDNA synthesis, 2 ml of the reaction mixture was utilized for PCR. 3. Results and discussion 3.1. EIAV NCp11 is thermostable EIAV NCp11 was expressed in E. coli BL21 (DE3). The 16 kDa IPTG-induced NCp11, which can be recognized by the anti-NCp11 antibody (Supplementary Fig. S1), was detected in the soluble fraction (S0) almost exclusively (Fig. 1A). When the soluble E. coli lysate was incubated at 100 C for 5 (S5), 10 (S10), 30 (S30) or 60 (S60) min, respectively, and clarified, a considerable portion of NCp11 remained in the soluble fraction, whereas most bacterial proteins were not detected. Fusion with 6 histidine tag at the N-terminus
Fig. 2. NCp11 maintains its RNA-binding activity after heat treatment. A. 1 mg purified NCp11 was incubated at 100 C for 0, 5, 10, 30 or 60 min, clarified, and employed for in vitro binding with E. coli total RNA and EMSA (right). SDS-PAGE analysis of heat-treated NCp11 was also shown (left). B. Quantitative results of A with three repeats. The amounts of RNA bound to per mg heated NCp11 are shown. C. 5 mg purified NCp11 was heated as in A, clarified and quantified. 1 mg of each heated NCp11 was employed for in vitro RNA-binding and EMSA. D. Quantitative results of C with three repeats. E. E. coli BL21 (DE3) was transformed with pET28-NCp11 (lanes 3 and 6) or co-transformed with pET28-NCp11 and pBSEIAVpsi (lanes 1, 2, 4 and 5). Total RNA (lanes 1 and 4) or RNA extracted from purified NCp11-RNA complexes (lanes 2, 3, 5 and 6) were utilized as RT-PCR (lanes 1e3) or PCR (lanes 4e6) templates to amplify a 124 bp fragment containing EIAVpsi. F. The clarified cell lysate of the IPTG-induced co-transformed E. coli BL21 (DE3) were incubated at 100 C for 0, 5, 10, 30 or 60 min, respectively, before affinity chromatography. RNA was extracted from each of the purified NCp11-RNA complexes and RT-PCR was conducted as in E.
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allowed purification of NCp11 by affinity chromatography. When the purified NCp11 was heated at 100 C, 91% (5 min), 81% (10 min), 61% (30 min) and 43% (60 min) of the NCp11 remained soluble (Fig. 1B and C). To investigate the structural integrity, NCp11 was analyzed by circular dichroism (Fig. 1D). The CD spectrum of NCp11 is similar to that of HIV NCp7 [22,23], with a positive maximum at ~218 nm and a negative minimum at ~199 nm. When NCp11 was heated, decreasing in the negative ellipticity was detected. However, a small red shift of the minimum ellipticity, indicative of a more unordered structure [22], was not observed until NCp11 was heated at 100 C for 60 min, demonstrating that NCp11 is thermostable. As controls, if the purified NCp11 was incubated at lower temperatures for 5, 10, 30 or 60 min, the percentages of soluble NCp11 retained were 105%, 109%, 105% and 98% at 0 C, 102%, 102%, 98% and 95% at 37 C, 96%, 96%, 95% and 89% at 60 C, respectively (Fig. 1E and F), confirming that NCp11 is also stable at these temperatures. 3.2. Heat-treated NCp11 maintained its RNA-binding activity Since NC can bind to RNA either specifically or nonspecifically, we next asked whether incubation at 100 C influenced the RNAbinding activity of NCp11. The E. coli RNA was readily associated with NCp11 nonspecifically in the electrophoretic mobility shift assay (EMSA), and was retarded at the top of the 0.7% agarose gel
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(Fig. 2A and C). When 1 mg NCp11 was incubated at 100 C for different periods of time, clarified, and subjected to EMSA with 0.5 mg RNA, the retarded RNA at the top of the agarose gel decreased along with the decreasing of the soluble NCp11 (Fig. 2A). When quantified, however, the relative binding activities of NCp11 (the amounts of RNA bound to per mg NCp11), were basically unchanged (Fig. 2B). If the same amounts of heat-treated soluble NCp11 (1 mg) were employed for EMSA, similar amounts of RNA were retarded (Fig. 2C), and not surprisingly, the relative binding activities of NCp11 remained unchanged (Fig. 2D). These results demonstrated that heat treatment didn't affect the nonspecific RNA-binding activity of NCp11. Because in vitro specific binding of HIV NC to psi RNA has been reported [24], the effect of heat treatment of NCp11 on its EIAVpsibinding activity was investigated. The psi sequence of EIAV was not as well-characterized as that in HIV [25]. Therefore, sequences covering most of the LTR U5 region and part of the gag gene [17] were included in the pBS-EIAVpsi. E. coli BL21 (DE3) was transformed with pET28-NCp11 or co-transformed with pET28-NCp11 and pBS-EIAVpsi. Unless RNase A was utilized, NCp11 was purified as NCp11-RNA complex (Supplementary Figs. S2 and S3), which enabled us to detect the binding of NCp11 to EIAVpsi. The NCp11associated RNA was extracted, and RT-PCR was performed to amplify the EIAVpsi (Fig. 2E). The 124 bp RT-PCR products were detected when the template was total RNA (lane 1, Fig. 2E) or RNA
Fig. 3. The effect of heat treatment on the inhibition of the first-strand cDNA synthesis by NCp11. A. NCp11 inhibits RT-PCR. Purified NCp11-RNA complex (lane 2), or RNA isolated from the complex (lanes 1 and 3e5), were used in RT-PCR. 250 mM imidazole (lanes 3e5) and 341 ng purified NCp11 (lane 4) or EIAV p9 (lane 5) were added in the RT-PCR reaction mixtures. B. NCp11 inhibits first-strand cDNA synthesis. 341 ng purified NCp11 (lanes 2, 5, 8 and 11) or EIAV p9 (lanes 3, 6, 9 and 12) was added in the reaction mixtures. After reverse transcriptions, proteins were removed. DNase I was utilized (lanes 7e12), inactivated, and PCR (lanes 1e3 and 7e9) or another RT-PCR (lanes 4e6 and 10e12) were subsequently conducted. C. SDS-PAGE analysis of the purified EIAV reverse transcriptase. D. NCp11 inhibits the first-strand cDNA synthesis catalyzed by the EIAV reverse transcriptase. E. Heat-treated NCp11 can still inhibit the first-strand cDNA synthesis. NCp11 was incubated at 100 C for 0, 5, 10, 30 or 60 min and clarified. Equal amounts (341 ng) of each sample were analyzed by SDS-PAGE (lower) or added in RT-PCR catalyzed by either the commercial (upper) or the EIAV (middle) reverse transcriptase.
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extracted from the NCp11-RNA complex which was purified from the E. coli co-transformed with pET28-NCp11 and pBS-EIAVpsi (lane 2, Fig. 2E). However, if the template was RNA extracted from the complex purified from the E. coli transformed only with pET28NCp11, no RT-PCR product was detected (lane 3, Fig. 2E). By using any of the RNA templates, no PCR product was detected (lanes 4e6, Fig. 2E), eliminating of plasmid DNA contamination. These results demonstrated that NCp11 can bind to the EIAVpsi in E. coli. To investigate the effect of heat treatment on the NCp11-EIAVpsi binding activity, clarified cell lysate of the IPTG-induced co-transformed E. coli BL21 (DE3) was kept at 100 C for 0, 5, 10, 30 or 60 min, respectively, centrifuged and subsequently employed for affinity chromatography. RNA was extracted from each of the NCp11-RNA complexes and utilized for RT-PCR. As the result, the 124 bp products were detected for all the samples (lanes 1e5, Fig. 2F), suggesting that the NCp11 maintained its EIAVpsi-binding activity upon heat treatment.
3.3. Addition of NCp11 inhibited the first-strand cDNA synthesis and heat-treated NCp11 maintained its inhibition In the mature virus, NC molecules coat the viral gRNA extensively at a very high degree of sequence occupancy, and the gRNA is still coated when viral cDNA synthesis takes place in the reverse transcription complex in the host cell [7]. We next sought to investigate how NCp11 affects first-strand cDNA synthesis (Fig. 3A). As proved in Fig. 2E, the 124 bp RT-PCR product was detected by using the RNA template extracted from the NCp11-EIAVpsi complex (lane 1, Fig. 3A). However, no RT-PCR product was seen if the NCp11-EIAVpsi complex was directly used (lane 2, Fig. 3A). Consistently, if purified NCp11 was added to the RT-PCR reaction mixture at a NC:NA ratio of 1:2, no product was detected (lane 4, Fig. 3A). This was not due to the contamination of imidazole nor other nonspecific proteins, since addition of imidazole and EIAV p9 which was purified by the same method did not prevent the appearance of the RT-PCR products (lanes 3 and 5, Fig. 3A). These
Fig. 4. The effects of protein concentration, buffer pH and EDTA on the NCp11 thermostability. 0.1, 0.3 or 0.9 mg/ml NCp11 in PBS pH 7.4 (A), NCp11 at pH 8, 7, 6, 5 or 4 (B), or NCp11 dialyzed against PBS supplemented with 10 mM EDTA (C), were heated and detected by SDS-PAGE (left). Quantified results were also shown (right).
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results demonstrate that EIAV NCp11 can inhibit RT-PCR at a very high degree of template RNA occupancy. To distinguish whether NCp11 inhibits the first-strand cDNA synthesis or the PCR, NCp11 was removed by phenol extraction after reverse transcriptions (Fig. 3B). As the result, the 124 bp PCR product was still not detected (lane 2, Fig. 3B). However, if another RT-PCR was performed, the 124 bp product was detected (lane 5, Fig. 3B), suggesting that at very high degree of sequence occupancy, NCp11 proteins coat and protect the RNA template, prevent the first-strand cDNA synthesis. If the reversed transcribed first-strand cDNAs were removed by DNase I, the appearance of RT-PCR products (lanes 10e12, Fig. 3B), but not PCR products (lanes 7e9, Fig. 3B), confirmed that the 124 bp signals were resulted from the RNA templates, and NCp11 inhibited the first-strand cDNA synthesis. We next questioned whether NCp11 inhibits the first-strand cDNA synthesis catalyzed by EIAV reverse transcriptase. The EIAV reverse transcriptase, a heterodimer of p66 and p51, was prepared (Fig. 3C). As expected, the EIAV reverse transcriptase catalyzed the first-strand cDNA synthesis from the RNA template extracted from the NCp11-EIAVpsi complex, which was detected by PCR (lane 1, Fig. 3D). Again, if the NCp11-EIAVpsi complex was utilized directly or if the purified NCp11 was supplemented into the reaction mixture, no products were detected (lanes 2 and 4, Fig. 3D), confirming that NCp11 inhibited the first-strand cDNA synthesis catalyzed by EIAV reverse transcriptase. To investigate the effect of heat treatment of NCp11 on the inhibition of the first-strand cDNA synthesis, equal amounts of heattreated and clarified NCp11 (lower) were added in each of the RTPCR reaction mixtures, catalyzed by either the commercial (upper) or the EIAV (middle) reverse transcriptase (Fig. 3E). It turned out that no RT-PCR products were detected after NCp11 was incubated at 100 C for 0, 5, 10, 30 or 60 min, demonstrating that heattreated NCp11 maintained its ability to inhibit the first-strand cDNA synthesis at the NC:NA ratio of 1:2. It has been reported that in the process of cDNA elongation, the NC protein, with its nucleic acid chaperone activity, helps to destabilize RNA secondary structures that might impede the reverse transcriptase movement across the genome. However, there are conflicting reports as to whether NC has an effect on the reverse transcriptase processivity [9]. Here we showed that at the NC:NA ratio of 1:2, NCp11 inhibited first-strand cDNA synthesis. Actually, at a NC:NA ratio of 1:10, NCp11 still inhibited first-strand cDNA synthesis, although not completely, however, at a NC:NA ratio of 1:50, NCp11 could barely inhibit anymore (Supplementary Fig. S4). In the viral replication cycle, the degree of sequence occupancy by NC is considered as dynamic [7], which may affects not only NC-mediated NA condensing and chaperoning activity, but also the roles that NC played in the first-strand cDNA synthesis. 3.4. The effects of protein concentration, buffer pH and EDTA on the NCp11 thermostability To investigate whether protein concentration affects the NCp11 thermostability, purified NCp11 was adjusted to 0.1, 0.3 or 0.9 mg/ml, respectively, and subjected to 100 C incubations. The retained percentages of soluble NCp11 were almost the same (Fig. 4A), suggesting that protein concentrations, at the range from 0.1 to 0.9 mg/ml, have not affected the NCp11 thermostability significantly. To investigate the potential roles that basic residues and zinc fingers played in the NCp11 thermostability, different buffer pH and 10 mM EDTA were utilized. At the range from pH 8 to 4, the retained soluble NCp11 increased along with the decreasing of buffer pH. At pH 5, 79% of the NCp11 remained soluble after heated at 100 C for 60 min, and at pH 4, almost all NCp11 remained soluble (Fig. 4B),
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suggesting that basic residues may be critical for the NCp11 thermostability. Addition of 10 mM EDTA to chelate zinc ions also slightly increased the thermostability of NCp11. About 93%, 89%, 82% or 59% of NCp11 remained soluble when it was incubated at 100 C for 5, 10, 30 or 60 min, respectively (Fig. 4C). In this study, we report that the EIAV NCp11 is thermostable. Upon heat treatment, NCp11 maintained its nonspecific RNA and the EIAVpsi-binding activities, maintained its inhibition of the firststrand cDNA synthesis at a very high degree of sequence occupancy. Currently, whether the thermostable property is unique to EIAV NCp11 or is universal for other lentiviral NC proteins are under investigation. During viral maturation, all the processes including the proteolytic cleavage of Gag precursors, the polymerization of CA to form the cone-shaped electron-dense core, and the condensation of the viral gRNA by NC are accompanied by vigorous energy exchange. What roles that NC thermostability played in the viral maturation process, and how it benefits the viral viability and infectivity awaits further investigation. Acknowledgements This work was supported by the Natural Science Foundation of China (No. 81470095 and No. 31870158) and the Natural Science Foundation of Tianjin City (No. 12JCYBJC15200, 16JCYBJC24000). We thank Jing Nan for technical assistance with the circular dichroism analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.137. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.137. References [1] J.L. Darlix, M. Lapadat-Tapolsky, H. de Rocquigny, B.P. Roques, First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses, J. Mol. Biol. 254 (1995) 523e537. [2] P. Amodeo, M.A.C. Morelli, A. Ostuni, G. Battistuzzi, A. Bavoso, Structural features in EIAV NCp11: a lentivirus nucleocapsid protein with a short linker, Biochemistry 45 (2006) 5517e5526. [3] J.A. Briggs, H.G. Krausslich, The molecular architecture of HIV, J. Mol. Biol. 410 (2011) 491e500. [4] M. Kuzembayeva, K. Dilley, L. Sardo, W.S. Hu, Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles, Virology 454e455 (2014) 362e370. [5] J. Kafaie, R. Song, L. Abrahamyan, A.J. Mouland, M. Laughrea, Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging, Virology 375 (2008) 592e610. [6] K. Wiegers, G. Rutter, H. Kottler, U. Tessmer, H. Hohenberg, H.G. Krausslich, Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites, J. Virol. 72 (1998) 2846e2854. [7] J.-L. Darlix, J. Godet, R. Ivanyi-Nagy, P. Fosse, O. Mauffret, Y. Mely, Flexible nature and specific functions of the HIV-1 nucleocapsid protein, J. Mol. Biol. 410 (2011) 565e581. [8] C. Vuilleumier, E. Bombarda, N. Morellet, D. Gerard, B.P. Roques, Y. Mely, Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study, Biochemistry 38 (1999) 16816e16825. [9] J.G. Levin, J. Guo, I. Rouzina, K. Musier-Forsyth, Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism, Prog. Nucleic Acid Res. Mol. Biol. 80 (2005) 217e286. [10] A. Rein, L.E. Henderson, J.G. Levin, Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication, Trends Biochem. Sci. 23 (1998) 297e301. [11] S. Carteau, R.J. Gorelick, F.D. Bushman, Coupled integration of human immunodeficiency virus type 1 cDNA ends by purified integrase in vitro: stimulation by the viral nucleocapsid protein, J. Virol. 73 (1999) 6670e6679. [12] M. Mori, L. Kovalenko, S. Lyonnais, D. Antaki, B.E. Torbett, M. Botta,
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1883e1889. [20] M. Bohlooli, A.A. Moosavi-Movahedi, F. Taghavi, P. Maghami, A.A. Saboury, Z. Moosavi-Movahedi, M. Farhadi, J. Hong, N. Sheibani, M. Habibi-Rezaei, Investigation of thermal reversibility and stability of glycated human serum albumin, Int. J. Biol. Macromol. 62 (2013) 358e364. [21] B.W. Ahn, D.U. Song, Y.D. Jung, K.O. Chay, M.A. Chung, S.Y. Yang, B.A. Shin, Detection of beta-amyloid peptide aggregation using DNA electrophoresis, Anal. Biochem. 284 (2000) 401e405. [22] J.G. Omichinski, G.M. Clore, K. Sakaguchi, E. Appella, A.M. Gronenborn, Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy, FEBS Lett. 292 (1991) 25e30. [23] Q.A. de Paula, J.B. Mangrum, N.P. Farrell, Zinc finger proteins as templates for metal ion exchange: substitution effects on the C-finger of HIV nucleocapsid NCp7 using M(chelate) species (M¼Pt, Pd, Au), J. Inorg. Biochem. 103 (2009) 1347e1354. [24] J. Dannull, A. Surovoy, G. Jung, K. Moelling, Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues, EMBO J. 13 (1994) 1525e1533. [25] S.C. Keane, X. Heng, K. Lu, S. Kharytonchyk, V. Ramakrishnan, G. Carter, S. Barton, A. Hosic, A. Florwick, J. Santos, N.C. Bolden, S. McCowin, D.A. Case, B.A. Johnson, M. Salemi, A. Telesnitsky, M.F. Summers, RNA structure. Structure of the HIV-1 RNA packaging signal, Science 348 (2015) 917e921.
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Thermostable properties of the equine infectious anemia virus nucleocapsid protein NCp11 Jinzhong Wang a, b, c, Qinghua Wang a, Shasha Hao a, Chao Guo a, Jing An a, Qingmiao Zhang a, Ruonan Liang a, Ying Wang a, b, c, * a b c
TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, 300457, China Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, 23 Hongda Street, TEDA, Tianjin, 300457, China Tianjin Key Laboratory of Microbial Functional Genomics, 23 Hongda Street, TEDA, Tianjin, 300457, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 January 2019 Accepted 31 January 2019 Available online 5 February 2019
Retroviral nucleocapsid (NC) proteins are multifunctional nucleic acid binding proteins, playing critical roles in essentially every step of the viral replication cycle. As a small, basic protein, NC contains one or two highly conserved zinc-finger domains, each having an invariant CCHC motif, flanked by basic residues. In this study, we report for the first time, to our knowledge, the thermostable property of equine infectious anemia virus (EIAV) NCp11. About 43% of purified NCp11 remained soluble after incubation at 100 C for 60 min, and heat-treated NCp11 maintained its abilities to bind to the E. coli RNA and the EIAV packaging signal sequence. At a very high degree of sequence occupancy, NCp11 inhibited first-strand cDNA synthesis catalyzed by either a commercial or the purified EIAV reverse transcriptase, and heattreated NCp11 still inhibited the first-strand cDNA synthesis. We also found that protein concentrations, at a range from 0.1 to 0.9 mg/ml, have not affected the NCp11 thermostability significantly. However, NCp11 at acidic pH was more thermostable. Our findings highlight a new feature of the NC protein. Detailed understanding of NC's properties and functions will facilitate the development of effective and rational therapeutic strategies against retroviruses. © 2019 Elsevier Inc. All rights reserved.
Keywords: EIAV Nucleocapsid NCp11 Thermostable RNA binding First-strand cDNA synthesis
1. Introduction Retroviral nucleocapsid (NC) proteins are small, highly basic proteins with one or two zinc finger domains, each containing the invariant CCHC Zn2þ ion binding motif, flanked by basic residues [1,2]. The mature NC protein is derived from the Gag polyprotein which is composed of matrix, capsid, NC domains and a C-terminal peptide [3]. The NC domain is responsible for the recognition of viral packaging signal (psi, j), resulting in the specific encapsidation of two copies of genomic RNA (gRNA) into the viral particle [4]. Some mutations affecting the NC domain of human immunodeficiency virus (HIV) Gag reduce the virus infectivity dramatically [5]. During virion maturation, Gag polyproteins are cleaved by virus-encoded protease in a highly-regulated and well-ordered manner to generate viral structure proteins [6]. Among them, about 1500e2000 NC molecules are associated with the viral RNA,
* Corresponding author. TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin, 300457, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.bbrc.2019.01.137 0006-291X/© 2019 Elsevier Inc. All rights reserved.
coating and protecting the entire dimeric gRNA in a histone-like manner [7]. NC protein can bind to different nucleic acid (NA) sequences at varying affinities [8], and is regarded as an ATPindependent NA chaperone [9,10]. At low degree of sequence occupancy when the NC:NA ratio is about 1:100 nt, NC binds specific viral sequences with high affinity; At high degree when the NC:NA ratio is from 1:15 to 1:7, NC binds NAs, causing their aggregation and driving the remodeling of the NC-NA complexes to reach compact and stable structures; At very high degree when NC:NA ratio is from 1:5 to 1:1, NC coats NAs extensively, providing protection to, or even freezes the compact nucleoprotein complexes [7]. Upon entry into the host cell, the NA chaperone activity of HIV NC is critical for its regulation of the reverse transcription. NC stabilizes interactions between tRNA primer and gRNA template, increases the efficiency of minus-strand transfer, helps the reverse transcriptase to destabilize RNA secondary structure, and contributes to the overall plus-strand transfer [9]. Besides, HIV NC also stimulates the integration of cDNA in vitro [11].
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As a multifunctional nucleic acid binding protein playing critical roles in essentially every step of the viral replication cycle, NC has been regarded as an ideal target for antiviral therapy [12], and has attracted more and more attention. Equine infectious anaemia virus (EIAV) was the first-reported lentivirus and retrovirus [13]. Compared with other lentiviruses, EIAV has the smallest genome size, encodes fewer regulatory proteins, and the first successful lentiviral vaccine was developed for EIAV [13,14]. In this study, the EIAV NCp11 was investigated, and for the first time, to our knowledge, the thermostability of NCp11 was found. Heat-treated NCp11 maintained its abilities to bind to E. coli RNA or EIAVpsi, maintained its ability to inhibit the first-strand cDNA synthesis catalyzed by either a commercial or the EIAV reverse transcriptase. Detailed understanding of NC's properties will benefit the understanding of its functions in viral replication and facilitate the development of effective and rational therapeutic strategies against retroviruses. 2. Materials and methods 2.1. Plasmids Plasmids pET28-NCp11 and pET28-p9 were described previously [15]. Plasmid pBS-EIAVpsi was constructed, which contains the EIAVuk genome (AF016316.1) from nt 287 to nt 546 covering the EIAVpsi sequence [16,17], using primers listed in the Supplementary. Coding regions of the EIAV reverse transcriptase p66 and p51 were amplified and cloned into pET32 and pET28 vectors, respectively. All plasmids constructed in this study were confirmed by DNA sequencing. 2.2. Protein purification, heat treatment and SDS-PAGE E. coli BL21 (DE3) harboring pET28-NCp11 or pET28-p9 was
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induced with 0.1 mM isopropylthio-b-D-galactoside (IPTG) for 4 h at 37 C, and that harboring pET32-EIAVp66 or pET28EIAVp51 was induced with 0.05 mM IPTG for 6 h at room temperature. 6 histidine-tagged proteins were purified as described [15]. In some experiments, before or after the cell lysate was employed onto the HIS-Select® Nickel Affinity Gel (Sigma-Aldrich), 20 mg/ml RNase A was added and samples were incubated on ice or at 37 C for 30e60 min. Purified NCp11, p9 or the equimolar mixture of p66 and p51 were dialyzed overnight against phosphate-buffered saline (PBS) unless otherwise specified. Protein concentrations were determined by bicinchoninic acid assay (Thermo). To determine the thermostability of NCp11, purified NCp11 or sonicated clear cell lysate of IPTG-induced E. coli BL21 (DE3) was incubated at 100 C for 0, 5, 10, 30 or 60 min, respectively, and then centrifuged to remove precipitated proteins. Purified NCp11 at different concentrations (0.1, 0.3 or 0.9 mg/ml), and NCp11 dialyzed against 20 mM acetate buffer (pH 5 or 4), phosphate buffer (pH 8, 7 or 6) [18] or PBS supplemented with 10 mM EDTA were also heated. SDS-PAGE was performed as described [15]. The levels of soluble NCp11 retained were quantified using the ImageJ software and normalized to the NCp11 level without heating [19]. 2.3. The far UV circular dichroism (CD) spectroscopic analysis Purified NCp11 was heated, clarified, adjusted to 0.2 mg/ml with PBS. Protein samples were transferred into a quartz glass cuvette with a 1 mm path length to a Jasco J-715 spectropolarimeter (Jasco, Japan) which was purged continuously with nitrogen, and scanned at 50 nm/min at 25 C over a wavelength range of 190e260 nm. Results of the CD measurements were converted to the mean residue ellipticities q [20].
Fig. 1. NCp11 is thermostable. A. E. coli BL21 (DE3) transformed with pET28-NCp11 was uninduced (lane 2) or induced with 0.1 mM IPTG for 4 h (lanes 3e8). Total (lanes 2e3) and soluble proteins heated at 100 C for 0 (lane 4), 5 (lane 5), 10 (lane 6), 30 (lane 7) or 60 min (lane 8) were analyzed by 12% SDS-PAGE. B. Purified NCp11 was incubated at 100 C for 0 (lane 2), 5 (lane 3), 10 (lane 4), 30 (lane 5) or 60 min (lane 6) and analyzed by SDS-PAGE. C. Quantitative results of B with three repeats. D. Circular dichroism spectra of NCp11 with or without heat treatment. E. Purified NCp11 was incubated at 0 C, 37 C or 60 C, respectively, and analyzed by SDS-PAGE. F. Quantitative measurements of E.
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2.4. In vitroRNA binding and electrophoretic mobility shift assay (EMSA) Total RNA was extracted from E. coli BL21 (DE3) using the Ultrapure RNA Kit (CoWin Biosciences), and quantified with NanoDrop ND-1000. For in vitro NCp11-RNA binding experiments, binding buffer (10 mM Hepes, pH 7.9, 50 mM potassium chloride, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM magnesium chloride and 0.5 mM PMSF) was used, and reaction mixtures were incubated at room temperature for 10 min. Samples were analyzed by EMSA [21] using 0.7% agarose gel in 1 TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) containing 0.1 mg/ml ethidium bromide. Images were acquired with the UV transilluminator, and shifted bands were quantified with the ImageJ software. 2.5. PCR and RT-PCR E. coli BL21 (DE3) was transformed with pET28-NCp11 or cotransformed with pET28-NCp11 and pBS-EIAVpsi. NCp11-RNA complexes were purified by nickel affinity chromatography, and RNA in the complexes was extracted. RT-PCR or PCR was performed using the SuperRT One Step RT-PCR Kit or the 2 Es Taq Master Mix (CoWin Biosciences), amplifying a 124 bp fragment (nt 338 to nt
461 of EIAVuk). Primers are listed in the Supplementary. For some experiments, 0.5 U Recombinant DNase I (RNase-free) and 5 U RNase Inhibitor (TaKaRa) were utilized to remove the first-strand cDNA. If the EIAV reverse transcriptase was used, 10 ml reaction mixture containing 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.2 mM dNTPs, 20 U RNase Inhibitor, 1 mM of each primer, 50 ng purified p66/p51 heterodimer and 40 ng template RNA was incubated at 37 C for 30 min. After first-strand cDNA synthesis, 2 ml of the reaction mixture was utilized for PCR. 3. Results and discussion 3.1. EIAV NCp11 is thermostable EIAV NCp11 was expressed in E. coli BL21 (DE3). The 16 kDa IPTG-induced NCp11, which can be recognized by the anti-NCp11 antibody (Supplementary Fig. S1), was detected in the soluble fraction (S0) almost exclusively (Fig. 1A). When the soluble E. coli lysate was incubated at 100 C for 5 (S5), 10 (S10), 30 (S30) or 60 (S60) min, respectively, and clarified, a considerable portion of NCp11 remained in the soluble fraction, whereas most bacterial proteins were not detected. Fusion with 6 histidine tag at the N-terminus
Fig. 2. NCp11 maintains its RNA-binding activity after heat treatment. A. 1 mg purified NCp11 was incubated at 100 C for 0, 5, 10, 30 or 60 min, clarified, and employed for in vitro binding with E. coli total RNA and EMSA (right). SDS-PAGE analysis of heat-treated NCp11 was also shown (left). B. Quantitative results of A with three repeats. The amounts of RNA bound to per mg heated NCp11 are shown. C. 5 mg purified NCp11 was heated as in A, clarified and quantified. 1 mg of each heated NCp11 was employed for in vitro RNA-binding and EMSA. D. Quantitative results of C with three repeats. E. E. coli BL21 (DE3) was transformed with pET28-NCp11 (lanes 3 and 6) or co-transformed with pET28-NCp11 and pBSEIAVpsi (lanes 1, 2, 4 and 5). Total RNA (lanes 1 and 4) or RNA extracted from purified NCp11-RNA complexes (lanes 2, 3, 5 and 6) were utilized as RT-PCR (lanes 1e3) or PCR (lanes 4e6) templates to amplify a 124 bp fragment containing EIAVpsi. F. The clarified cell lysate of the IPTG-induced co-transformed E. coli BL21 (DE3) were incubated at 100 C for 0, 5, 10, 30 or 60 min, respectively, before affinity chromatography. RNA was extracted from each of the purified NCp11-RNA complexes and RT-PCR was conducted as in E.
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allowed purification of NCp11 by affinity chromatography. When the purified NCp11 was heated at 100 C, 91% (5 min), 81% (10 min), 61% (30 min) and 43% (60 min) of the NCp11 remained soluble (Fig. 1B and C). To investigate the structural integrity, NCp11 was analyzed by circular dichroism (Fig. 1D). The CD spectrum of NCp11 is similar to that of HIV NCp7 [22,23], with a positive maximum at ~218 nm and a negative minimum at ~199 nm. When NCp11 was heated, decreasing in the negative ellipticity was detected. However, a small red shift of the minimum ellipticity, indicative of a more unordered structure [22], was not observed until NCp11 was heated at 100 C for 60 min, demonstrating that NCp11 is thermostable. As controls, if the purified NCp11 was incubated at lower temperatures for 5, 10, 30 or 60 min, the percentages of soluble NCp11 retained were 105%, 109%, 105% and 98% at 0 C, 102%, 102%, 98% and 95% at 37 C, 96%, 96%, 95% and 89% at 60 C, respectively (Fig. 1E and F), confirming that NCp11 is also stable at these temperatures. 3.2. Heat-treated NCp11 maintained its RNA-binding activity Since NC can bind to RNA either specifically or nonspecifically, we next asked whether incubation at 100 C influenced the RNAbinding activity of NCp11. The E. coli RNA was readily associated with NCp11 nonspecifically in the electrophoretic mobility shift assay (EMSA), and was retarded at the top of the 0.7% agarose gel
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(Fig. 2A and C). When 1 mg NCp11 was incubated at 100 C for different periods of time, clarified, and subjected to EMSA with 0.5 mg RNA, the retarded RNA at the top of the agarose gel decreased along with the decreasing of the soluble NCp11 (Fig. 2A). When quantified, however, the relative binding activities of NCp11 (the amounts of RNA bound to per mg NCp11), were basically unchanged (Fig. 2B). If the same amounts of heat-treated soluble NCp11 (1 mg) were employed for EMSA, similar amounts of RNA were retarded (Fig. 2C), and not surprisingly, the relative binding activities of NCp11 remained unchanged (Fig. 2D). These results demonstrated that heat treatment didn't affect the nonspecific RNA-binding activity of NCp11. Because in vitro specific binding of HIV NC to psi RNA has been reported [24], the effect of heat treatment of NCp11 on its EIAVpsibinding activity was investigated. The psi sequence of EIAV was not as well-characterized as that in HIV [25]. Therefore, sequences covering most of the LTR U5 region and part of the gag gene [17] were included in the pBS-EIAVpsi. E. coli BL21 (DE3) was transformed with pET28-NCp11 or co-transformed with pET28-NCp11 and pBS-EIAVpsi. Unless RNase A was utilized, NCp11 was purified as NCp11-RNA complex (Supplementary Figs. S2 and S3), which enabled us to detect the binding of NCp11 to EIAVpsi. The NCp11associated RNA was extracted, and RT-PCR was performed to amplify the EIAVpsi (Fig. 2E). The 124 bp RT-PCR products were detected when the template was total RNA (lane 1, Fig. 2E) or RNA
Fig. 3. The effect of heat treatment on the inhibition of the first-strand cDNA synthesis by NCp11. A. NCp11 inhibits RT-PCR. Purified NCp11-RNA complex (lane 2), or RNA isolated from the complex (lanes 1 and 3e5), were used in RT-PCR. 250 mM imidazole (lanes 3e5) and 341 ng purified NCp11 (lane 4) or EIAV p9 (lane 5) were added in the RT-PCR reaction mixtures. B. NCp11 inhibits first-strand cDNA synthesis. 341 ng purified NCp11 (lanes 2, 5, 8 and 11) or EIAV p9 (lanes 3, 6, 9 and 12) was added in the reaction mixtures. After reverse transcriptions, proteins were removed. DNase I was utilized (lanes 7e12), inactivated, and PCR (lanes 1e3 and 7e9) or another RT-PCR (lanes 4e6 and 10e12) were subsequently conducted. C. SDS-PAGE analysis of the purified EIAV reverse transcriptase. D. NCp11 inhibits the first-strand cDNA synthesis catalyzed by the EIAV reverse transcriptase. E. Heat-treated NCp11 can still inhibit the first-strand cDNA synthesis. NCp11 was incubated at 100 C for 0, 5, 10, 30 or 60 min and clarified. Equal amounts (341 ng) of each sample were analyzed by SDS-PAGE (lower) or added in RT-PCR catalyzed by either the commercial (upper) or the EIAV (middle) reverse transcriptase.
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extracted from the NCp11-RNA complex which was purified from the E. coli co-transformed with pET28-NCp11 and pBS-EIAVpsi (lane 2, Fig. 2E). However, if the template was RNA extracted from the complex purified from the E. coli transformed only with pET28NCp11, no RT-PCR product was detected (lane 3, Fig. 2E). By using any of the RNA templates, no PCR product was detected (lanes 4e6, Fig. 2E), eliminating of plasmid DNA contamination. These results demonstrated that NCp11 can bind to the EIAVpsi in E. coli. To investigate the effect of heat treatment on the NCp11-EIAVpsi binding activity, clarified cell lysate of the IPTG-induced co-transformed E. coli BL21 (DE3) was kept at 100 C for 0, 5, 10, 30 or 60 min, respectively, centrifuged and subsequently employed for affinity chromatography. RNA was extracted from each of the NCp11-RNA complexes and utilized for RT-PCR. As the result, the 124 bp products were detected for all the samples (lanes 1e5, Fig. 2F), suggesting that the NCp11 maintained its EIAVpsi-binding activity upon heat treatment.
3.3. Addition of NCp11 inhibited the first-strand cDNA synthesis and heat-treated NCp11 maintained its inhibition In the mature virus, NC molecules coat the viral gRNA extensively at a very high degree of sequence occupancy, and the gRNA is still coated when viral cDNA synthesis takes place in the reverse transcription complex in the host cell [7]. We next sought to investigate how NCp11 affects first-strand cDNA synthesis (Fig. 3A). As proved in Fig. 2E, the 124 bp RT-PCR product was detected by using the RNA template extracted from the NCp11-EIAVpsi complex (lane 1, Fig. 3A). However, no RT-PCR product was seen if the NCp11-EIAVpsi complex was directly used (lane 2, Fig. 3A). Consistently, if purified NCp11 was added to the RT-PCR reaction mixture at a NC:NA ratio of 1:2, no product was detected (lane 4, Fig. 3A). This was not due to the contamination of imidazole nor other nonspecific proteins, since addition of imidazole and EIAV p9 which was purified by the same method did not prevent the appearance of the RT-PCR products (lanes 3 and 5, Fig. 3A). These
Fig. 4. The effects of protein concentration, buffer pH and EDTA on the NCp11 thermostability. 0.1, 0.3 or 0.9 mg/ml NCp11 in PBS pH 7.4 (A), NCp11 at pH 8, 7, 6, 5 or 4 (B), or NCp11 dialyzed against PBS supplemented with 10 mM EDTA (C), were heated and detected by SDS-PAGE (left). Quantified results were also shown (right).
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results demonstrate that EIAV NCp11 can inhibit RT-PCR at a very high degree of template RNA occupancy. To distinguish whether NCp11 inhibits the first-strand cDNA synthesis or the PCR, NCp11 was removed by phenol extraction after reverse transcriptions (Fig. 3B). As the result, the 124 bp PCR product was still not detected (lane 2, Fig. 3B). However, if another RT-PCR was performed, the 124 bp product was detected (lane 5, Fig. 3B), suggesting that at very high degree of sequence occupancy, NCp11 proteins coat and protect the RNA template, prevent the first-strand cDNA synthesis. If the reversed transcribed first-strand cDNAs were removed by DNase I, the appearance of RT-PCR products (lanes 10e12, Fig. 3B), but not PCR products (lanes 7e9, Fig. 3B), confirmed that the 124 bp signals were resulted from the RNA templates, and NCp11 inhibited the first-strand cDNA synthesis. We next questioned whether NCp11 inhibits the first-strand cDNA synthesis catalyzed by EIAV reverse transcriptase. The EIAV reverse transcriptase, a heterodimer of p66 and p51, was prepared (Fig. 3C). As expected, the EIAV reverse transcriptase catalyzed the first-strand cDNA synthesis from the RNA template extracted from the NCp11-EIAVpsi complex, which was detected by PCR (lane 1, Fig. 3D). Again, if the NCp11-EIAVpsi complex was utilized directly or if the purified NCp11 was supplemented into the reaction mixture, no products were detected (lanes 2 and 4, Fig. 3D), confirming that NCp11 inhibited the first-strand cDNA synthesis catalyzed by EIAV reverse transcriptase. To investigate the effect of heat treatment of NCp11 on the inhibition of the first-strand cDNA synthesis, equal amounts of heattreated and clarified NCp11 (lower) were added in each of the RTPCR reaction mixtures, catalyzed by either the commercial (upper) or the EIAV (middle) reverse transcriptase (Fig. 3E). It turned out that no RT-PCR products were detected after NCp11 was incubated at 100 C for 0, 5, 10, 30 or 60 min, demonstrating that heattreated NCp11 maintained its ability to inhibit the first-strand cDNA synthesis at the NC:NA ratio of 1:2. It has been reported that in the process of cDNA elongation, the NC protein, with its nucleic acid chaperone activity, helps to destabilize RNA secondary structures that might impede the reverse transcriptase movement across the genome. However, there are conflicting reports as to whether NC has an effect on the reverse transcriptase processivity [9]. Here we showed that at the NC:NA ratio of 1:2, NCp11 inhibited first-strand cDNA synthesis. Actually, at a NC:NA ratio of 1:10, NCp11 still inhibited first-strand cDNA synthesis, although not completely, however, at a NC:NA ratio of 1:50, NCp11 could barely inhibit anymore (Supplementary Fig. S4). In the viral replication cycle, the degree of sequence occupancy by NC is considered as dynamic [7], which may affects not only NC-mediated NA condensing and chaperoning activity, but also the roles that NC played in the first-strand cDNA synthesis. 3.4. The effects of protein concentration, buffer pH and EDTA on the NCp11 thermostability To investigate whether protein concentration affects the NCp11 thermostability, purified NCp11 was adjusted to 0.1, 0.3 or 0.9 mg/ml, respectively, and subjected to 100 C incubations. The retained percentages of soluble NCp11 were almost the same (Fig. 4A), suggesting that protein concentrations, at the range from 0.1 to 0.9 mg/ml, have not affected the NCp11 thermostability significantly. To investigate the potential roles that basic residues and zinc fingers played in the NCp11 thermostability, different buffer pH and 10 mM EDTA were utilized. At the range from pH 8 to 4, the retained soluble NCp11 increased along with the decreasing of buffer pH. At pH 5, 79% of the NCp11 remained soluble after heated at 100 C for 60 min, and at pH 4, almost all NCp11 remained soluble (Fig. 4B),
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suggesting that basic residues may be critical for the NCp11 thermostability. Addition of 10 mM EDTA to chelate zinc ions also slightly increased the thermostability of NCp11. About 93%, 89%, 82% or 59% of NCp11 remained soluble when it was incubated at 100 C for 5, 10, 30 or 60 min, respectively (Fig. 4C). In this study, we report that the EIAV NCp11 is thermostable. Upon heat treatment, NCp11 maintained its nonspecific RNA and the EIAVpsi-binding activities, maintained its inhibition of the firststrand cDNA synthesis at a very high degree of sequence occupancy. Currently, whether the thermostable property is unique to EIAV NCp11 or is universal for other lentiviral NC proteins are under investigation. During viral maturation, all the processes including the proteolytic cleavage of Gag precursors, the polymerization of CA to form the cone-shaped electron-dense core, and the condensation of the viral gRNA by NC are accompanied by vigorous energy exchange. What roles that NC thermostability played in the viral maturation process, and how it benefits the viral viability and infectivity awaits further investigation. Acknowledgements This work was supported by the Natural Science Foundation of China (No. 81470095 and No. 31870158) and the Natural Science Foundation of Tianjin City (No. 12JCYBJC15200, 16JCYBJC24000). We thank Jing Nan for technical assistance with the circular dichroism analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.137. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.137. References [1] J.L. Darlix, M. Lapadat-Tapolsky, H. de Rocquigny, B.P. Roques, First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses, J. Mol. Biol. 254 (1995) 523e537. [2] P. Amodeo, M.A.C. Morelli, A. Ostuni, G. Battistuzzi, A. Bavoso, Structural features in EIAV NCp11: a lentivirus nucleocapsid protein with a short linker, Biochemistry 45 (2006) 5517e5526. [3] J.A. Briggs, H.G. Krausslich, The molecular architecture of HIV, J. Mol. Biol. 410 (2011) 491e500. [4] M. Kuzembayeva, K. Dilley, L. Sardo, W.S. Hu, Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles, Virology 454e455 (2014) 362e370. [5] J. Kafaie, R. Song, L. Abrahamyan, A.J. Mouland, M. Laughrea, Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging, Virology 375 (2008) 592e610. [6] K. Wiegers, G. Rutter, H. Kottler, U. Tessmer, H. Hohenberg, H.G. Krausslich, Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites, J. Virol. 72 (1998) 2846e2854. [7] J.-L. Darlix, J. Godet, R. Ivanyi-Nagy, P. Fosse, O. Mauffret, Y. Mely, Flexible nature and specific functions of the HIV-1 nucleocapsid protein, J. Mol. Biol. 410 (2011) 565e581. [8] C. Vuilleumier, E. Bombarda, N. Morellet, D. Gerard, B.P. Roques, Y. Mely, Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study, Biochemistry 38 (1999) 16816e16825. [9] J.G. Levin, J. Guo, I. Rouzina, K. Musier-Forsyth, Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism, Prog. Nucleic Acid Res. Mol. Biol. 80 (2005) 217e286. [10] A. Rein, L.E. Henderson, J.G. Levin, Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication, Trends Biochem. Sci. 23 (1998) 297e301. [11] S. Carteau, R.J. Gorelick, F.D. Bushman, Coupled integration of human immunodeficiency virus type 1 cDNA ends by purified integrase in vitro: stimulation by the viral nucleocapsid protein, J. Virol. 73 (1999) 6670e6679. [12] M. Mori, L. Kovalenko, S. Lyonnais, D. Antaki, B.E. Torbett, M. Botta,
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