Protein Expression and Purication 93 (2014) 3237

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Protein Expression and Purication

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Expression, purication, and characterization of full-length bovine leukemia virus Gag protein from bacterial culture
Dominic F. Qualley , Bethany L. Boleratz
Department of Chemistry, Berry College, Mt. Berry, GA 30149, United States

a r t i c l e

i n f o

a b s t r a c t
In retroviruses, the Gag protein is a precursor from which the mature proteins matrix, capsid, and nucleocapsid are derived. Gag plays an important structural role in the assembly of virions at the plasma membrane. While Gag proteins from several different retroviruses have been puried for study in vitro, there has yet to be a report of successful purication of deltaretroviral Gag. In this paper, we report the cloning, expression and purication of full-length bovine leukemia virus (BLV) Gag from Escherichia coli using a combination of polyethyleneimine precipitation, ammonium sulfate precipitation, and afnity chromatography. Experiments using size-exclusion chromatography were also performed to analyze the oligomeric state of the Gag protein in solution, and results suggest that it exists primarily as a monomer but may oligomerize into higher-order complexes to a small extent. Molecular weight estimation by comparison of elution volume to a set of protein standards supports the hypothesis that BLV Gag adopts a slightly extended conformation in solution. The results are discussed in comparison to the solution structure and assembly pathways of other retrovirus genera. 2013 Elsevier Inc. All rights reserved.

Article history: Received 2 October 2013 and in revised form 10 October 2013 Available online 23 October 2013 Keywords: Retrovirus Gag Size-exclusion chromatography Viral assembly Bovine leukemia virus Matrix Nucleocapsid

Introduction Retroviruses are a family of viruses that use a virally-encoded reverse transcriptase to convert their (+)-single stranded RNA genomes into double-stranded DNA prior to nuclear import and integration into the host cells genome [1]. There are three genes common to all retroviruses: gag, pol, and env. The gag gene encodes the structural proteins of the virus; these include but are not limited to matrix (MA),1 capsid (CA), and nucleocapsid (NC). During replication, these proteins are expressed as a single polypeptide, simply referred to as Gag. After assembly and release from the cell, Gag is proteolyzed into its constituent proteins in a process known as maturation. Prior to release of the immature virion from the cell, the Gag protein plays an important role in viral assembly and genome packaging [27]. In human immunodeciency virus type 1 (HIV-1), a well-characterized retrovirus, the NC domain of Gag strongly binds to the genomic RNA while the MA domain binds to the plasma membrane of the cell through both electrostatic interactions and insertion of a covalently-attached myristoyl group into the lipid bilayer. The CA domain facilitates GagGag interactions, promoting multimerization at the inner leaet of the membrane [810].

Corresponding author. Tel.: +1 706 368 5718.

E-mail address: dqualley@berry.edu (D.F. Qualley). Abbreviations used: MA, matrix; CA, capsid; NC, nucleocapsid; FIV, feline immunodeciency virus; HTLV-1, human T-cell leukemia virus type one; VLPs, virus-like particles; IPTG, isopropyl beta-D-1-thiogalactopyranoside.
1

While MA, CA, and NC have been successfully expressed and puried as individual proteins from a variety of different retroviruses, expression and purication of full-length Gag has proven to be much more challenging. This presents a signicant roadblock for progress in understanding how retroviral assembly works, since pure protein is essential for biophysical studies that examine structure, binding, and oligomerization. HIV-1 Gag used for in vitro experiments typically contains several key mutations to preserve solubility and stability. First, the C-terminal peptide (p6) that exists immediately following the NC domain is deleted (commonly referred to as a Dp6 mutant). Compared to wild-type HIV-1 Gag, GagDp6 is degraded to a much lesser extent when expressed in bacterial culture [11]. GagDp6 can be puried to 8590% homogeneity and with much better yields than the wild-type protein. Second, two mutations (W316A and M317A) are made to adjacent residues in the CA domain. This mutant, termed WM-Gag, has been shown to be predominantly monomeric in solution whereas the wild-type protein is primarily dimeric [12]. A third variant contains the Dp6 mutation as well as another deletion, D1699, which has been shown to enhance in vitro assembly of Gag; the purication of this construct has been reported in great detail [13]. Interestingly, full-length Gag from feline immunodeciency virus (FIV) has been successfully puried using afnity chromatography without any modications, save the 6 His tag [14]. This result is surprising, since HIV-1 and FIV are both lentiviruses and their Gag proteins share sequence homology.

1046-5928/$ - see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2013.10.008

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Several Gag proteins from other retrovirus genera have been puried from bacterial culture and used for in vitro experiments. Murine leukemia virus (MLV, a gammaretrovirus) Gag was isolated and its properties in solution were shown to differ signicantly from what has been observed for HIV-1 Gag [15]. While evidence exists that HIV-1 Gag adopts a compact conformation [12,16,17], small-angle X-ray scattering experiments show that MLV Gag has a rigid, extended structure in solution. Wild-type MLV Gag is also predominately monomeric, while HIV-1 Gag exists in a monomerdimer equilibrium. Gag protein from an alpharetrovirus, Rous sarcoma virus (RSV) has been puried in its native form with the exception of the C-terminal protease domain, which is normally translated as part of the Gag polyprotein in RSV [18]. This led to work showing that RSV Gag required nucleic acid to promote dimer formation as an intermediate step in assembly [19,20]. To date, successful purication of deltaretroviral Gag has not been reported. The most notable human deltaretrovirus is human T-cell leukemia virus type one (HTLV-1). HTLV-1 is endemic in many areas of the world, and is the causative agent of adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis [21]. Cell culture experiments have shown that, as for other retroviruses, elements of HTLV-1 Gag are critical for membrane binding and genome packaging [22,23]. Interestingly, there are some key differences between HIV-1 and HTLV-1 in terms of the behavior of Gag in solution; HTLV-1 Gag is primarily monomeric, and forms virus-like particles (VLPs) with a poorly organized Gag lattice compared to HIV-1 [2426]. Bovine leukemia virus (BLV), another deltaretrovirus, infects domestic cattle and is prevalent in dairy herds in the United States [27]. Although humans cannot be infected with BLV, infected cattle have shown reduced milk production compared to healthy animals [28], making study of BLV important from an agricultural standpoint. Additionally, antibodies reactive to BLV capsid have been discovered in human blood sera, presumably due to consumption of products from infected animals [29]. Finally, BLV may also be useful as an animal model for development of a vaccine for HTLV-1 due to the similarity of BLV and HTLV-1 [30,31]. Previous work has shown that BLV Gag relies on both its MA and NC domains for effective genome packaging; zinc-binding residues in the NC domain and basic residues in the MA domain appear to be especially important [32]. Furthermore, myristylation of the N-terminal glycine residue of Gag and the presence of a PPPY motif were also shown to be crucial for the production of VLPs [33]. While the role of the myristate moiety in this context is clear, less is known about the interaction of BLV Gag with the plasma membrane of the host cell. Both MA [34] and NC [35] of BLV have been puried and used in biophysical experiments that characterize nucleic acid binding, but the use of authentic Gag would provide a clearer and more accurate representation of the events that occur during replication. In this paper, we report the rst successful purication of a fulllength deltaretroviral Gag protein. Cloning, expression, and purication procedures are described in detail, along with preliminary experiments designed to characterize the properties of BLV Gag in solution. Our results show that Gag has been puried to near homogeneity, and that it exists in solution as a partially-extended monomer.

previously described construct [36]. The forward primer contained an XhoI restriction site at the 50 -end of the sequence coding for Gag, while the reverse primer contained an AvrII site positioned after the stop codon at the 30 -end. The sequences of the primers were as follows: 50 -GTGACACTCGAGATGGGAAATTCCCCC-30 (forward) and 50 -GTGACACCTAGGTTAGTTTTTTGATTTGAGGG-30 (reverse). Both primers were purchased from Integrated DNA Technologies (Coralville, IA) and used without further purication. Following PCR amplication, the DNA product and the expression vector pET45b (EMD Millipore, Darmstadt, Germany) were each digested with XhoI and AvrII (New England Biolabs, Ipswich, MA) using standard molecular biology protocols. The digestion reactions were cleaned up using a PureLink PCR Purication kit (Life Technologies, Grand Island, NY); the High-Cutoff binding buffer provided was used to remove DNA less than 300 bp in length. The Gag DNA insert (25 ng) and linearized pET45b (20 ng) were incubated at 16 C for 4 h in the presence of 2 Weiss units of T4 DNA ligase (EMD Millipore) in a total volume of 10 lL according to the manufacturers instructions. The entire ligation reaction was transformed into chemically competent NovaBlue Escherichia coli cells (EMD Millipore). Several colonies were screened for the presence of BLV Gag DNA by colony PCR; colonies that were positive were grown in liquid culture and plasmids were puried using a commercially available miniprep kit (Omega Bio-Tek, Norcross, GA). The presence of BLV Gag DNA was conrmed by restriction digests of the puried plasmids, and plasmids were sequenced to conrm that the correct sequence had been inserted. The expression plasmid was then transformed into chemically competent Rosetta-2 (DE3) pLysS cells (EMD Millipore), a strain that contains a plasmid encoding a number of tRNA genes corresponding to rare codons. Protein expression and purication A small culture (10 mL) of LB media containing 100 lg/mL ampicillin and 34 lg/mL chloramphenicol was inoculated with a single colony of Rosetta-2 (DE3) pLysS cells containing the BLV Gag expression plasmid. After shaking overnight at 37 C, the small culture was added to 500 mL of LB media (with antibiotics at the concentrations used for the small culture) and incubated at 37 C with agitation until the optical density of the culture at 600 nm reached 0.5. At this point, the culture was allowed to cool to room temperature before addition of isopropyl beta-D-1-thiogalactopyranoside (IPTG) to a nal concentration of 1 mM. Expression was allowed to continue overnight at room temperature with shaking, and cultures were harvested by centrifugation for 20 min at 8000 rpm. Cell pellets were stored at 80 C until purication took place. Cell pellets (about 2.4 g wet weight, representing 500 mL of liquid culture) were thawed on ice and resuspended completely in 20 mL of lysis buffer (50 mM HEPES pH 7.0, 0.3 M NaCl, 1 mM tris (2-carboxyethyl) phosphineHCl, 10% glycerol, 10 mM imidazole, and 1 mM phenylmethylsulfonyl uoride). The cells were sonicated on ice using 10 cycles of the following sequence: 1 s on, 1 s off for 20 s; rest on ice for 40 s. Polyethyleneimine was then added to a nal concentration of 0.15%, and the lysate was allowed to incubate on ice for 15 min to precipitate nucleic acids. After incubation, a thick white precipitate was observed. The lysate was centrifuged at 11,000 rpm for 20 min, and the pellet discarded. To the supernatant was added 1/2 volume of ammonium sulfate solution (saturated at room temperature), dropwise, on ice, with stirring. Following addition, the solution was allowed to incubate on ice for 30 min without stirring. The solution was then centrifuged at 11,000 rpm for 20 min. The supernatant from this step was discarded, and the solid white pellet was gently and completely resuspended on ice with 1 mL of column buffer (50 mM HEPES pH 7.0,

Materials and methods Cloning The DNA sequence corresponding to full-length Gag was amplied by PCR using a plasmid template (pKB426, a kind gift from Dr. Kathleen Boris-Lawrie) which is a derivative of pBLV-SVNEO, a

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0.3 M NaCl, 1 mM tris (2-carboxyethyl) phosphineHCl, and 10% glycerol). The solution was added to a column containing 1 mL of cOmplete His-Tag Purication Resin (Roche Applied Science, Indianapolis, IN) and washed extensively with column buffer containing 10 mM imidazole. BLV Gag was eluted using a step gradient of imidazole in column buffer. Three elution buffers (10 mL each, containing 50, 200, and 500 mM imidazole) were added sequentially to the column, and the eluate was collected in 5 mL fractions. Fractions were run on a 12% SDSPAGE gel, where the majority of pure Gag protein was found to elute at 200 mM imidazole. The Gag-containing fractions were pooled and concentrated to a volume of about 200 lL using an Amicon centrifugal ltration device with a 30 kDa molecular weight cut-off (EMD Millipore); the protein was also exchanged into column buffer (without imidazole) at this time. The concentration of puried BLV Gag was determined using UV absorbance at 280 nm with an extinction coefcient of 79,800 M1 cm1, calculated from the amino acid sequence as described previously [37]. The measured A260/A280 ratio was about 0.5, indicating that the protein was free of contaminating nucleic acids. Zinc acetate was added (2.2 equiv., 1.1 per zinc nger), and the Gag protein was stored in aliquots at 80 C until further use. Size-exclusion chromatography Size-exclusion experiments were performed using a Yarra SEC2000 HPLC column (Phenomenex, Torrance, CA) with a length of 300 mm and an internal diameter of 4.6 mm. The column was connected to a PerkinElmer Series 200 HPLC system with the detector set to 280 nm. For the molecular weight standards and for BLV Gag, a 50 lL sample was injected and eluted at a ow rate of 0.50 mL/ min with a buffer containing 50 mM sodium phosphate pH 7.0 and 150 mM NaCl. The void volume (V0) of the column was determined by injection of a 1 mg/mL solution of blue dextran while monitoring the absorbance at 360 nm. The column was calibrated by sequential injections of 1 mg/mL solutions of the proteins listed in Table 1. A calibration curve was constructed by plotting the ratio of elution volume to void volume (Ve/V0) of each standard vs. log molecular weight, and an equation for the curve was determined by linear regression. Results and discussion Expression and purication of retroviral Gag from bacterial culture can be a surprisingly difcult endeavor. We initially tried to express BLV Gag in two other expression vectors (pET15b and pET32a); however, despite trying a wide variety of expression conditions, the protein was only expressed as a truncation product (pET32a) or was not expressed at all (pET15b). Next, we attempted to express Gag using the vector pET45b, which would produce full-

Fig. 1. 1% agarose gel run in TAE buffer showing the successful amplication of the Gag gene from the pKB426 template. The approximate size of the PCR product (on the left) corresponds to the projected size of 1203 bp.

length Gag with a 6 His-tag at the N-terminus. The Gag DNA was amplied with primers designed to insert an XhoI restriction site at the 50 -end of the sequence and an AvrII site at the 30 -end. The size of the PCR product corresponded with the expected size (1203 nucleotides, Fig. 1). Following digestion, ligation, and sequencing, the plasmid was transformed into Rosetta-2 (DE3) pLysS cells for expression. Initially, expression was induced at 37 C and was allowed to con-

Table 1 Proteins run as standards on Yarra SEC-2000 size-exclusion column. The molecular weights and retention times of the standards were used to construct the calibration curve for BLV Gag size estimation (Fig. 3). Blue dextran was used to determine the void volume of the column. Protein Blue dextran b-Glucosidase Amyloglucosidase Bovine serum albumin Ovalbumin Horseradish peroxidase Myoglobin a-Lactalbumin Cytochrome c Molecular weight (kDa) 2000 135 72 66.5 44.3 44.0 17.7 14.2 12.3 Elution volume (mL) 2.08 2.72 2.87 2.85 3.05 3.03 3.41 3.40 3.50

Fig. 2. Samples from Gag purication run on SDSPAGE (12%). Lane 1 is a protein standard (Precision Plus, Bio-Rad, Hercules, CA) with the molecular weight of each standard listed in kDa on the left. Lane 2 is the supernatant from the ammonium sulfate precipitation. Lanes 310 represent fractions from the Ni2+ afnity chromatography, consisting of resuspended ammonium sulfate pellet loaded onto the column (3), eluate from wash step (4), eluate using 50 mM imidazole (5 and 6), eluate using 200 mM imidazole (7 and 8), and eluate using 500 mM imidazole (9 and 10).

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tinue for 4 h, with time point being drawn from the culture before induction and every hour post-induction. SDSPAGE analysis of the samples indicated that very little protein was being expressed over time; the protein that was overexpressed was much smaller than the expected BLV Gag. The concentration of IPTG used for induction was varied from 0.1 to 4 mM with no discernable difference in expression level. When induction was carried out at room temperature and allowed to proceed overnight, there were bands corresponding to molecular weights of about 50 kDa (theoretical MW of our Gag construct = 47.7 kDa) and about 16 kDa. When purication was attempted, the smaller product co-puried with the larger product, leading to the conclusion that the smaller protein was a

Fig. 3. Calibration curve of size-exclusion column used to estimate molecular weight of BLV Gag. Identities and molecular weights of the standards are listed in Table 1. The triangle data point, identied with an arrow, indicates the position of BLV Gag on the calibration curve when its theoretical molecular weight (47.7 kDa) is used.

truncated version of the full-length Gag. Due to this observation, we inserted an ammonium sulfate precipitation step immediately prior to column purication. Addition of ammonium sulfate to up to 40% saturation has been used in the purication of other Gag proteins, and was sufcient to precipitate Gag in all reported cases [13,15,18]. For BLV Gag, addition of ammonium sulfate to 33% saturation (achieved by addition of 1/2 volume of saturated ammonium sulfate solution prepared at room temperature) followed by a brief incubation period on ice adequately precipitated the desired protein from solution. After the suspension was centrifuged and the pellet resuspended in a minimal volume of column buffer, Ni2+-afnity chromatography yielded highly puried BLV Gag (Fig. 2). The size and shape of BLV Gag in solution was investigated using size-exclusion chromatography. Eight protein standards were run (Table 1) and their elution volumes plotted against the log molecular weight to generate a standard curve (Fig. 3). BLV Gag was injected at a concentration of 5 lM, and the elution prole was obtained by plotting A280 as a function of time (Fig. 4). The chromatogram shows a large peak centered around 5.80 min, which corresponds to a molecular weight of about 67.4 kDa. This is signicantly larger than 47.7 kDa, the calculated molecular weight of puried BLV Gag. Some peak tailing was observed, possibly due to a small amount of conformational heterogeneity of protein in the sample. Peak tailing is also often associated with non-specic interactions between the analyte and stationary phase, but in this case is unlikely given the high salt concentration used in the running buffer. A much smaller peak is seen at 4.54 min, corresponding to a molecular weight of about 420 kDa. This observation has two potential implications. First, BLV Gag exists predominately as a monomer at concentrations below 5 lM. This is not surprising, given the dissociation constants of other retroviral Gag proteins (5 lM for HIV-1 and 400 lM for MLV) [12,15]. Additionally, uorescence uctuation spectroscopy experiments using HeLa cells expressing HTLV-1 Gag failed to detect any signicant Gag dimerization in the cytoplasm [25]. Second, the shape of BLV Gag in solution most likely approximates a partially extended protein. A protein existing in a rigid, extended conforma-

Fig. 4. Size exclusion chromatography elution prole of BLV Gag when injected as a 5 lM solution. Absorbance at 280 nm was monitored during elution at 0.5 mL/min.

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D.F. Qualley, B.L. Boleratz / Protein Expression and Purication 93 (2014) 3237 [12] S.A. Datta, Z. Zhao, P.K. Clark, S. Tarasov, J.N. Alexandratos, S.J. Campbell, M. Kvaratskhelia, J. Lebowitz, A. Rein, Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch, J. Mol. Biol. 365 (2007) 799811. [13] S.A. Datta, A. Rein, Preparation of recombinant HIV-1 gag protein and assembly of virus-like particles in vitro, Methods Mol. Biol. 485 (2009) 197208. [14] J.L. Affranchino, S.A. Gonzalez, In vitro assembly of the feline immunodeciency virus Gag polyprotein, Virus Res. 150 (2010) 153157. [15] S.A. Datta, X. Zuo, P.K. Clark, S.J. Campbell, Y.X. Wang, A. Rein, Solution properties of murine leukemia virus gag protein: differences from HIV-1 gag, J. Virol. 85 (2011) 1273312741. [16] S.A. Datta, J.E. Curtis, W. Ratcliff, P.K. Clark, R.M. Crist, J. Lebowitz, S. Krueger, A. Rein, Conformation of the HIV-1 Gag protein in solution, J. Mol. Biol. 365 (2007) 812824. [17] S.A. Datta, F. Heinrich, S. Raghunandan, S. Krueger, J.E. Curtis, A. Rein, H. Nanda, HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid, J. Mol. Biol. 406 (2011) 205214. [18] S. Campbell, V.M. Vogt, In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identication of the p10 domain as a morphological determinant in the formation of spherical particles, J. Virol. 71 (1997) 44254435. [19] Y.M. Ma, V.M. Vogt, Rous sarcoma virus Gag proteinoligonucleotide interaction suggests a critical role for protein dimer formation in assembly, J. Virol. 76 (2002) 54525462. [20] Y.M. Ma, V.M. Vogt, Nucleic acid binding-induced Gag dimerization in the assembly of Rous sarcoma virus particles in vitro, J. Virol. 78 (2004) 5260. [21] L.B. Cook, M. Elemans, A.G. Rowan, B. Asquith, HTLV-1: persistence and pathogenesis, Virology 435 (2013) 131140. [22] H. Wang, N.J. Machesky, L.M. Mansky, Both the PPPY and PTAP motifs are involved in human T-cell leukemia virus type 1 particle release, J. Virol. 78 (2004) 15031512. [23] J. Inlora, V. Chukkapalli, D. Derse, A. Ono, Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)bisphosphate than those mediated by the matrix domain of HIV-1 Gag, J. Virol. 85 (2011) 38023810. [24] I.F. Grigsby, W. Zhang, J.L. Johnson, K.H. Fogarty, Y. Chen, J.M. Rawson, A.J. Crosby, J.D. Mueller, L.M. Mansky, Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry, Retrovirology 7 (2010) 75. [25] K.H. Fogarty, Y. Chen, I.F. Grigsby, P.J. Macdonald, E.M. Smith, J.L. Johnson, J.M. Rawson, L.M. Mansky, J.D. Mueller, Characterization of cytoplasmic GagGag interactions by dual-color z-scan uorescence uctuation spectroscopy, Biophys. J. 100 (2011) 15871595. [26] K.H. Fogarty, W. Zhang, I.F. Grigsby, J.L. Johnson, Y. Chen, J.D. Mueller, L.M. Mansky, New insights into HTLV-1 particle structure, assembly, and GagGag interactions in living cells, Viruses 3 (2011) 770793. [27] NAHMS-USDA, Bovine leukosis virus (BLV) on U.S. Dairy Operations, 2007. [28] S.L. Ott, R. Johnson, S.J. Wells, Association between bovine-leukosis virus seroprevalence and herd-level productivity on US dairy farms, Prev. Vet. Med. 61 (2003) 249262. [29] G.C. Buehring, S.M. Philpott, K.Y. Choi, Humans have antibodies reactive with Bovine leukemia virus, AIDS Res. Hum. Retroviruses 19 (2003) 11051113. [30] S.M. Rodriguez, A. Florins, N. Gillet, A. de Brogniez, M.T. Sanchez-Alcaraz, M. Boxus, F. Boulanger, G. Gutierrez, K. Trono, I. Alvarez, L. Vagnoni, L. Willems, Preventive and therapeutic strategies for bovine leukemia virus: lessons for HTLV, Viruses 3 (2011) 12101248. [31] N. Gillet, A. Florins, M. Boxus, C. Burteau, A. Nigro, F. Vandermeers, H. Balon, A.B. Bouzar, J. Defoiche, A. Burny, M. Reichert, R. Kettmann, L. Willems, Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human, Retrovirology 4 (2007) 18. [32] H. Wang, K.M. Norris, L.M. Mansky, Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging, J. Virol. 77 (2003) 94319438. [33] H. Wang, K.M. Norris, L.M. Mansky, Analysis of bovine leukemia virus gag membrane targeting and late domain function, J. Virol. 76 (2002) 84858493. [34] D.F. Qualley, C.M. Lackey, J.P. Paterson, Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: implications for deltaretroviral assembly, Proteins 81 (2013) 13771385. [35] D.R. Morcock, S. Katakam, B.P. Kane, J.R. Casas-Finet, Fluorescence and nucleic acid binding properties of bovine leukemia virus nucleocapsid protein, Biophys. Chem. 97 (2002) 203212. [36] D. Derse, L. Martarano, Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity, J. Virol. 64 (1990) 401405. [37] S.C. Gill, P.H. von Hippel, Calculation of protein extinction coefcients from amino acid sequence data, Anal. Biochem. 182 (1989) 319326. [38] V. Chukkapalli, A. Ono, Molecular determinants that regulate plasma membrane association of HIV-1 Gag, J. Mol. Biol. 410 (2011) 512524. [39] W. Zhou, L.J. Parent, J.W. Wills, M.D. Resh, Identication of a membranebinding domain within the amino-terminal region of human immunodeciency virus type 1 Gag protein which interacts with acidic phospholipids, J. Virol. 68 (1994) 25562569. [40] P. Spearman, J.J. Wang, N. Vander Heyden, L. Ratner, Identication of human immunodeciency virus type 1 Gag protein domains essential to membrane binding and particle assembly, J. Virol. 68 (1994) 32323242.

tion would elute much earlier than expected, as observed for MLV Gag. MLV Gag eluted at a volume that corresponds to over twice its calculated molecular weight, which was explained by small angle X-ray scattering experiments showing that MLV Gag exists in an elongated form [15]. This observation could have potential implications for the packaging of genomic RNA during replication; HIV-1 Gag is much more exible, and likely adopts a bent conformation, allowing both the MA and NC domains to bind the RNA [12,3845]. Although more evidence is needed to propose that BLV assembly follows a similar mechanism, puried BLV MA has been shown to bind both viral RNA sequences and inositol hexakisphosphate, which suggests such a mechanism could be plausible [34]. Although neither the structure of BLV CA nor NC has been solved, the CANC domain of BLV Gag is signicantly enriched in Pro residues, which could cause the polyprotein to remain inexible compared to HIV-1 Gag. As determined by NMR, the shape of BLV MA is globular, unlike that of MLV MA [46]. The identity of the protein eluting in the rst peak shown in Fig. 4 is much less clear. While it may be tempting to conclude that this peak is composed of aggregated BLV Gag, this is unlikely since the peak elutes later than the void volume. The molecular weight that was calculated based on the elution volume (420 kDa) is outside of the separation range of the column (1300 kDa); thus, 420 kDa can only be considered a rough estimate at best. However, it is interesting that the molecular weight of the early eluting peak corresponds roughly to that of a BLV Gag hexamer. It has been shown that retroviruses from different genera (HIV-1, RSV, MasonPzer monkey virus, and xenotropic murine leukemia virusrelated virus) assemble from hexameric Gag lattices based upon the N-terminal domain of CA [4749]. Although the presence of nucleic acid is required for VLP assembly, it is possible that BLV Gag is pre-assembling into hexamers prior to RNA-triggered VLP formation. Our laboratory is currently working on experiments that should clarify this issue further. Acknowledgments We are grateful to Dr. Kathleen Boris-Lawrie for provision of pKB426, the National Science Foundation (Major Research Instrumentation Grant CHE-1125616), and Berry College (Faculty Development Grant 2013-14-FDG-006). We also thank Drs. Karin Musier-Forsyth, Hally Shaffer, and Alice Suroviec for critical reading of the manuscript. References
[1] J.M. Cofn, S.H. Hughes, H. Varmus, Retroviruses, Cold Spring Harbor Laboratory Press, Plainview, NY, 1997. [2] V. DSouza, M.F. Summers, How retroviruses select their genomes, Nat. Rev. Microbiol. 3 (2005) 643655. [3] B.K. Ganser-Pornillos, M. Yeager, W.I. Sundquist, The structural biology of HIV assembly, Curr. Opin. Struct. Biol. 18 (2008) 203217. [4] E. Hamard-Peron, D. Muriaux, Retroviral matrix and lipids, the intimate interaction, Retrovirology 8 (2011) 15. [5] A. Rein, S.A. Datta, C.P. Jones, K. Musier-Forsyth, Diverse interactions of retroviral Gag proteins with RNAs, Trends Biochem. Sci. 36 (2011) 373380. [6] A.G. Bukrinskaya, HIV-1 assembly and maturation, Arch. Virol. 149 (2004) 10671082. [7] J.A. Webb, C.P. Jones, L.J. Parent, I. Rouzina, K. Musier-Forsyth, Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging, RNA (New York, NY) 19 (2013) 10781088. [8] D. Ako-Adjei, M.C. Johnson, V.M. Vogt, The retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles, J. Virol. 79 (2005) 1346313472. [9] C. Momany, L.C. Kovari, A.J. Prongay, W. Keller, R.K. Gitti, B.M. Lee, A.E. Gorbalenya, L. Tong, J. McClure, L.S. Ehrlich, M.F. Summers, C. Carter, M.G. Rossmann, Crystal structure of dimeric HIV-1 capsid protein, Nat. Struct. Biol. 3 (1996) 763770. [10] B.K. Ganser, S. Li, V.Y. Klishko, J.T. Finch, W.I. Sundquist, Assembly and analysis of conical models for the HIV-1 core, Science 283 (1999) 8083. [11] S. Campbell, A. Rein, In vitro assembly properties of human immunodeciency virus type 1 Gag protein lacking the p6 domain, J. Virol. 73 (1999) 22702279.

D.F. Qualley, B.L. Boleratz / Protein Expression and Purication 93 (2014) 3237 [41] A. Ono, S.D. Ablan, S.J. Lockett, K. Nagashima, E.O. Freed, Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane, Proc. Natl. Acad. Sci. USA 101 (2004) 1488914894. [42] A. Ono, E.O. Freed, Binding of human immunodeciency virus type 1 Gag to membrane: role of the matrix amino terminus, J. Virol. 73 (1999) 41364144. [43] J.S. Saad, J. Miller, J. Tai, A. Kim, R.H. Ghanam, M.F. Summers, Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly, Proc. Natl. Acad. Sci. USA 103 (2006) 1136411369. [44] P. Purohit, S. Dupont, M. Stevenson, M.R. Green, Sequence-specic interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection, RNA (New York, N.Y.) 7 (2001) 576584. [45] C.P. Jones, S.A. Datta, A. Rein, I. Rouzina, K. Musier-Forsyth, Matrix domain modulates HIV-1 Gags nucleic acid chaperone activity via inositol phosphate binding, J. Virol. 85 (2010) 15941603.

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[46] S. Matthews, M. Mikhailov, A. Burny, P. Roy, The solution structure of the bovine leukaemia virus matrix protein and similarity with lentiviral matrix proteins, EMBO J. 15 (1996) 32673274. [47] A. de Marco, N.E. Davey, P. Ulbrich, J.M. Phillips, V. Lux, J.D. Riches, T. Fuzik, T. Ruml, H.G. Krausslich, V.M. Vogt, J.A. Briggs, Conserved and variable features of Gag structure and arrangement in immature retrovirus particles, J. Virol. 84 (2010) 1172911736. [48] M. Yeager, E.M. Wilson-Kubalek, S.G. Weiner, P.O. Brown, A. Rein, Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms, Proc. Natl. Acad. Sci. USA 95 (1998) 72997304. [49] R. Hadravova, A. de Marco, P. Ulbrich, J. Stokrova, M. Dolezal, I. Pichova, T. Ruml, J.A. Briggs, M. Rumlova, In vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus XMRV, J. Virol. 86 (2012) 1297 1306.