Conserved cysteines in Mason–Pfizer monkey virus capsid protein are essential for infectious mature particle formation

Virology 521 (2018) 108–117

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Conserved cysteines in Mason–Pfizer monkey virus capsid protein are essential for infectious mature particle formation

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Růžena Píchalováa, Tibor Füzika,1, Barbora Vokatáa, Michaela Rumlováb, Manuel Llanoc, ⁎ Alžběta Dostálkováb, Ivana Křížováb, Tomáš Rumla, Pavel Ulbricha, a

Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague, Czech Republic Department of Biotechnology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague, Czech Republic c Department of Biological Sciences, University of Texas at El Paso, 500 West University El Paso, TX 79902, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: M-PMV capsid Cysteine mutagenesis Retrovirus assembly Virus core stability M-PMV infectivity

Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein–protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.

1. Introduction

enzymes from the Pol precursor. This cleavage triggers the structural reorganization of the immature particle into the mature one, where MA remains bound to the viral membrane and CA forms a viral core enclosing the condensed NC–RNA complex with the viral enzymes reverse transcriptase and integrase (Welker et al., 2000). Retroviral capsid protein consists of two independently folded domains: an N-terminal (CA-NTD) and C-terminal (CA-CTD) domain that are connected by a short flexible linker. Despite the low sequence homology of CAs across different retroviruses, CA proteins exhibit strong tertiary structure conservation among most retroviral genera (Campos-Olivas et al., 2000; Gamble et al., 1997; Gitti et al., 1996; Jin et al., 1999; Khorasanizadeh et al., 1999; Macek et al., 2009; Mortuza et al., 2009). The N-terminal part of CA-NTD forms a β-hairpin followed by six or seven α-helices, depending on the type of retrovirus. CA-CTD is a small globular domain formed by four α-helices (Campos-Olivas et al., 2000; Gamble et al., 1997; Gitti et al., 1996; Jin et al., 1999; Khorasanizadeh et al., 1999; Macek et al., 2009; Mortuza et al., 2009). Recently, high-resolution structures of in vitro-assembled as well as native immature particles of HIV-1, Mason-Pfizer monkey virus (MPMV) and Rous sarcoma virus (RSV) have been solved by cryo-electron microscopy and confirmed the hexameric arrangement of CA lattice within the immature particle (Bharat et al., 2012; Schur et al., 2015a, 2015b). A comparison of quaternary CA structures in the three above-

The major retroviral structural polyprotein Gag plays a key role in the assembly of immature retroviral particles (Coffin et al., 1997). Its transition to the infectious, mature virion includes subsequent budding and maturation. Gag polyproteins of all orthoretroviruses invariably contain three major domains connected by flexible linkers. The Nterminal matrix (MA) domain directs Gag to the site of assembly and also to the site of interaction with the plasma membrane and enables incorporation of Env glycoprotein into the forming particle. The capsid (CA) domain drives Gag multimerization during the immature particle assembly and forms the core of the mature virus. The nucleocapsid (NC) domain is responsible for specific viral genomic RNA incorporation and facilitates the immature particle assembly (Alfadhli et al., 2009; Briggs and Kräusslich, 2011; Campbell and Vogt, 1997; Gross et al., 1997; Checkley et al., 2011; Kafaie et al., 2008; Murray et al., 2005; Zhang and Barklis, 1995). In HIV-1, CA is next to spacer peptide 1 (SP1) that stabilizes the immature CA–SP1 lattice (Datta et al., 2011; Schur et al., 2015b). In M-PMV, there is a spacer-like domain that is functionally similar to other retroviral spacer peptides and contributes to the assembly of immature virus-like particles (Bohmová et al., 2010). Maturation is initiated by the self-activation of viral protease, which cleaves Gag into the aforementioned proteins and also liberates viral

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Corresponding author. E-mail addresses: [email protected] (R. Píchalová), [email protected], [email protected] (T. Füzik), [email protected] (B. Vokatá), [email protected] (M. Rumlová), [email protected] (M. Llano), [email protected] (A. Dostálková), [email protected] (I. Křížová), [email protected] (T. Ruml), [email protected] (P. Ulbrich). 1 Structural Virology, Central European Institute of Technology, Masaryk University, Kamenice 753/5, 62500, Brno, Czech Republic. https://doi.org/10.1016/j.virol.2018.06.001 Received 9 March 2018; Received in revised form 31 May 2018; Accepted 1 June 2018 0042-6822/ © 2018 Published by Elsevier Inc.

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Sanger DNA sequencing. For the in vitro protein assembly studies, the M-PMV ΔProCANC DNA constructs were based on the pSIT vector, where the gene encoding CA and NC fusion protein lacking the Nterminal proline of CA (Rumlová-Kliková et al., 2000) is under the control of the T7 promoter. The pTriExT Met-CA M-PMV-derived constructs were based on the commercial pTriEx-4 plasmid (Merck, Germany). It carries the gene for the M-PMV CA protein (missing 5 Cterminal amino acids of CA to increase the protein expression in bacterial cells) under the control of the T7 promoter. The construct provides the CA product with cleavable His–Smt3 tag at its N-terminus. Due to the presence of a proline at the M-PMV CA N-terminus, an additional methionine was added at the CA N-terminus to enable His–Smt3 tag cleavage by the active fragment of Ulp1 protease (Mossessova and Lima, 2000). Point mutations of M-PMV CA cysteines C82, C193 and C213 and their combinations (namely C82S, C82I, C193A, C193S, C193I, C213A, C213S, C82S/C193S, C82S/C213S, C193S/C213S and C82S/C193S/C213S) were introduced using SLIM mutagenesis (Chiu et al., 2004). For in vivo studies, the M-PMV Env expression vector (pTMO) (Brody et al., 1994) and pSARM-EGFP vector containing the M-PMV provirus with enhanced green fluorescent protein (EGFP) replacing the env gene (Newman et al., 2006) were used. The desired mutations (C193A, C193S, C213A, C213S, C193S/C213S) were first introduced into the SacI–Eco72I fragment of the M-PMV genome in the helper vector MHelppUC19 (Bohmová et al., 2010; Křížová et al., 2012) using EMILI mutagenesis (Füzik et al., 2014). After verifying the DNA sequence, the SacI–Eco72I fragments of MHelppUC19 carrying cysteine mutations were inserted into pSARMEGFP. Further details of vector preparations and sequences of PCR primers can be obtained from the authors upon request.

mentioned viruses revealed that they share conserved CA-CTD arrangements, while the organization of CA-NTD substantially differs. Furthermore, CA–CA interactions in the immature Gag lattice differ from those in the mature one. The immature Gag lattice is stabilized by inter-hexameric CA-CTD–CA-CTD interactions; while in the mature core intra-hexameric CA-NTD–CA-NTD and CA-NTD–CA-CTD interactions predominate (Bharat et al., 2012; Schur et al., 2015b). The CA-CTD domain plays a key role in the assembly and stabilization of the immature particle (Bharat et al., 2012; Schur et al., 2015b). In MPMV, the RKK motif in CA-CTD contributes to the recruitment of genomic RNA into immature particles (Füzik et al., 2016). In addition, the second αhelices of HIV-1 CA-CTDs form a dimeric interaction interface between two adjacent hexamers (Schur et al., 2015b). Furthermore, the W184A and M185A mutations in the second α-helix block HIV-1 CA dimerization in vitro (Gamble et al., 1997), reduce Gag–Gag interactions in vitro (Burniston et al., 1999) and also lead to a significant reduction in immature particle production (von Schwedler et al., 2003). Detailed structural analysis of this important interface indicates that within the hexamer, contacts between adjacent CA-CTD monomers are mediated by a highly conserved region in CA-CTD, called the major homology region (MHR) (Mammano et al., 1994; Schur et al., 2015b). Other CA-CTD residues, whose mutations disrupt viral particle assembly (Chu et al., 2006; von Schwedler et al., 2003), lie close to the inter-protein interaction interface stabilizing the immature HIV-1 CA lattice (Schur et al., 2015b). Besides the inhibition of assembly, some mutations in CA could also alter the stability of the core (Forshey et al., 2002; von Schwedler et al., 2003), subsequently influencing the early post-entry steps and virus infectivity. In HIV-1 CA-NTD, the mutations E45A and E128A/R132A stabilize the core, whereas the mutations R18A/N21A, P38A and L136D are core destabilizing (Forshey et al., 2002; von Schwedler et al., 2003). Similarly, the mutations K170A, K203A and Q219A in HIV-1 CA-CTD significantly decrease core stability (Forshey et al., 2002; von Schwedler et al., 2003). With the exception of spumaretroviruses and alpharetroviruses, retroviral CA-CTDs contain a pair of highly conserved cysteine residues (see Fig. S1, Supplementary section) which are in close spatial proximity (CamposOlivas et al., 2000), and their mutations severely affect the viral life cycle (McDermott et al., 1996; Nath and Peterson, 2001). In freshly prepared HIV-1 virions isolated from the cell culture, these conserved cysteine residues (C198 and C218) seem to be reduced (McDermott et al., 1996). In contrast, they form an intra-molecular disulfide bond in the crystal structures of HIV-1 and EIAV CA-CTD (Gamble et al., 1997; Jin et al., 1999; Worthylake et al., 1999). The C198S mutation results in greatly reduced HIV-1 infectivity, although it did not affect the assembly and particle release of HIV-1, indicating a post-assembly block (i.e. altered core stability, or block of viral entry or post-entry processes) (McDermott et al., 1996). In contrast, the C218S mutation virtually eliminated the HIV-1 immature particle assembly (McDermott et al., 1996). All the above-mentioned mutants, with altered core stability, were also severely impaired in virus infectivity and reverse transcription (Forshey et al., 2002; McDermott et al., 1996; von Schwedler et al., 2003). In order to obtain more information on the role of the conserved cysteines in CA-CTDs, we determined the effect of their mutations on the M-PMV core assembly in vitro, and the viral life cycle and infectivity. Mutagenesis analysis is a convenient tool that complements the findings from structural studies to identify the amino acid residues necessary for efficient viral infection. Our data indicate that the invariant cysteine residues in the CA protein of M-PMV have a fundamental role in virion assembly and infectivity.

2.2. Bacterial expression and purification of ΔProCANC proteins Luria-Bertani medium containing ampicillin (100 μg/ml) was inoculated with E. coli BL21(DE3) cells carrying the appropriate DNA construct. When the optical density at 600 nm reached 0.4–0.6, the protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG; 0.4 mM, final concentration). The cells were harvested 4 h post-induction by centrifugation and stored at − 20 °C. Retroviral proteins were purified according to the previously described protocols (Ulbrich et al., 2006; Voráčková et al., 2011) with minor modifications. Briefly, a high salt extraction buffer was used to solubilize and release ΔProCANC protein from the cell lysate pellet. Due to the insolubility of mutant proteins, both mutant proteins as well as the wt ΔProCANC protein were denatured with 8 M urea, and after the dialysis into equilibration buffer (50 mM phosphate, 500 mM NaCl, 3 M urea, pH 7,5), all the required proteins were purified by immobilized metal ion affinity chromatography on a Zn2+-charged column. Bound proteins were eluted with 2 M NH4Cl in equilibration buffer. Purified wt/mutant proteins were renatured by three-step dialysis into the storage buffer (50 mM phosphate, 500 mM NaCl, 1 μM ZnSO4, 0.05% mercaptoethanol, pH 7.5) containing the following concentrations of urea (1 M, 0 M, 0 M), concentrated to ~ 1 mg/ml by ultra-filtration (Amicon® Ultra-15 Centrifugal Filter Unit Ultracel® 10 K; Millipore, Carrigtwohill, Ireland) and stored at − 20 °C. The protein concentration was determined using Bradford protein assay. 2.3. Bacterial expression and purification of Met-CA proteins Luria-Bertani medium containing ampicillin (100 μg/ml) and chloramphenicol (100 μg/ml) was inoculated with E. coli Tuner™(DE3) pLacI cells carrying the appropriate DNA construct. When the optical density at 600 nm reached 0.8–1, the protein expression was induced by the addition of IPTG (0.4 mM, final concentration). The cells were harvested 4 h post-induction by centrifugation and stored at − 20 °C. The bacterial pellet from 0.5 l of cell culture was resuspended in 15 ml of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, 5 mM

2. Materials and methods 2.1. DNA constructs All plasmids were propagated in E. coli DH5α cells and verified by 109

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et al., 2014; Wildová et al., 2008; von Schwedler et al., 1998). The Met–CA protein solution, supplemented with 200 μM CoCl2, was treated with E. coli methionine amino peptidase (EcMAP; home-made sources). The reaction mixture was incubated overnight at room temperature, and subsequently mixed with 250 mg/ml PEG 3000 in a 1:1 (v/v) ratio. The assembled cores were fixed with 1% glutaraldehyde in phosphatebuffered saline (PBS) for 5 min, negatively stained and analyzed using TEM.

Tris(2-carboxyethyl)phosphine (TCEP), pH 8.0) containing 1 mg/ml lysozyme, 0.5% (w/v) n-Octyl-β-D-thioglucopyranoside, 100 μg/ml phenylmethylsulfonyl fluoride (PMSF) and 25 U/ml TurboNuclease™, and the solution was incubated on a rotating mixer for 30 min at room temperature. The lysate was then centrifuged at 20,000 ×g, 4 °C for 10 min and the supernatant containing the protein of interest was loaded onto a HisTrap™ HP (5 ml) column (GE Healthcare, Uppsala, Sweden) equilibrated with lysis buffer. After two washing steps with 25 ml of lysis buffer, the bound His-Smt3 tagged protein was eluted with 20 ml of elution buffer E1 (50 mM Tris-HCl, 150 mM NaCl, 350 mM imidazole, 5 mM TCEP, pH 8.0). Elution fractions were collected and adjusted to contain 10 mM dithiothreitol (DTT) and 100 μg/ ml PMSF, and the active fragment of Ulp1 protease (Mossessova and Lima, 2000) (home-made sources) was added to remove His–Smt3 tag from the purified protein. The mixture was incubated overnight on a rotating mixer at room temperature. After clarification of the cleavage mixture (20,000 ×g, 4 °C for 10 min), the imidazole was removed using a HiPrep™ 26/10 Desalting column (GE Healthcare, Uppsala, Sweden) equilibrated with buffer E2 (50 mM Tris-HCl, 100 mM NaCl, 5 mM TCEP, pH 8.0). Cleaved His–Smt3 tag was removed from the protein mixture using metal affinity chromatography on a HisTrap™ HP (5 ml) column equilibrated with buffer E2. M-PMV Met–CA protein was released from the column during the washing step with 25 ml of buffer E2, concentrated to ~ 10–30 mg/ml by ultrafiltration (Amicon® Ultra15 Centrifugal Filter Unit Ultracel® 10 K; Millipore, Carringtwohill, Ireland) and stored at − 20 °C.

2.7. Cell growth and virus production HEK 293 T cells were grown in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Gibco) and 1% L-glutamine (PAA Laboratories, Linz, AT). Typically, HEK 293 T cells were plated at a density of 3·105 cells/ml 24 h before transfection and then were co-transfected with wt/mutant M-PMV-expression vector pSARM-EGFP and virus glycoprotein-expression vector pTMO (Brody et al., 1994) using polyethylenimine (Sigma-Aldrich, St. Louis, Macao), according to the manufacturer's instructions. Culture media containing virions were harvested 48 or 72 h post-transfection, filtered through a 0.45 µm filter and centrifuged through a 20% sucrose cushion for 1 h at 210,000 ×g, 4 °C. M-PMV proteins were detected by Western blot using rabbit anti-M-PMV CA polyclonal antibody. 2.8. Protein expression, radioactive labeling and quantification of virus particle release

2.4. Analysis of virus-like particle formation in E. coli At 72 h post-transfection, the HEK 293 T cells were starved for 30 min in methionine- and cysteine-deficient DMEM (Sigma-Aldrich, St. Louis, Macao) and pulse-labeled for 30 min with 250 μCi/ml of Tran35Slabel (M.G.P., Zlín, Czech Republic). Labeled cells were then chased for 16 h. Culture media from chased cells were collected and adjusted to contain 12.5 mM Tris-HCl (pH 8.0), 0.5% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS. The cells from pulse and pulse–chase experiments were washed with cold PBS, lysed in 1 ml of lysis buffer (25 mM Tris-HCl, 50 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, pH 8.0) on ice for 10 min and clarified by centrifugation at 16,000 ×g for 2 min. Then SDS was added to a final concentration of 0.1%. M-PMV proteins were immunoprecipitated from the cell lysates and culture media with a rabbit anti-M-PMV CA polyclonal antibody (1:400 dilution) and separated by SDS-PAGE. Radiolabeled proteins were visualized by a PharosFX™ Plus Molecular Imager (Bio-Rad). To quantify the virus particle release, radiolabeled protein bands of pulselabeled [35S]-Gag (Pr78) and pulse–chase-labeled virion-associated [35S]-CA (p27) proteins were quantitated using Fiji (ImageJ) software package (Schindelin et al., 2012). The released viral proteins were calculated as relative concentrations of CA correlated to the levels of intracellular Gag in individual samples.

To determine whether both wt and mutant ΔProCANC proteins formed virus-like particles (VLPs) inside bacterial cells during their production, 1 ml of cell culture 4 h post-induction was pelleted, and the cell pellet was resuspended in 300 μl of lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 1 mM EDTA, 1% (w/v) octylthioglucoside, 1 mg/ml lysozyme, pH 8.0). The suspension was incubated on a rotating mixer for 20 min at room temperature. During this cell lysis process, the assembled virus-like particles (VLPs) were released from bacterial cells and were subsequently analyzed by transmission electron microscopy (TEM) after their negative staining. 2.5. In vitro assembly of ΔProCANC The assembly of VLPs from purified ΔProCANC proteins was studied as described previously (Füzik et al., 2016; Hadravová et al., 2012; Ulbrich et al., 2006). Since the addition of nucleic acids that bind to the NC domain was previously shown to enhance the in vitro VLP assembly process efficiency (Ulbrich et al., 2006), we mixed 60 μg of the protein in storage buffer with bacteriophage MS2 genomic RNA (MS2 RNA; Sigma-Aldrich, USA) in a 10:1 (w/w) ratio of protein to nucleic acid in a final reaction volume of 100 μl. The mixture was dialyzed against the assembly buffer (50 mM Tris-HCl, 100 mM NaCl, 1 μM ZnSO4, pH 7.8) for 2 h at room temperature. When the effect of reducing conditions on the in vitro assembly of VLPs was studied, the protein in the storage buffer was mixed with bacteriophage λ genomic DNA (λ DNA; SigmaAldrich, USA) as described above, and 60 mM DTT was included in the reaction mixture and 20 mM DTT in the assembly buffer. All the in vitro immature-like particle assembly experiments were performed in triplicates.

2.9. Single-round infectivity assay The infectivity was determined as described previously (Dostálková et al., 2018). At 72 h post-transfection, the virus-containing culture media were collected, filtered through a 0.45 µm filter and incubated for 15 min with polybrene (Sigma-Aldrich) at a final concentration of 1 mg/ml. Each sample was normalized for CA content by ELISA. The volume of culture media containing virions used to infect HEK 293 T cells was adjusted so that each sample contained an equivalent amount of CA. At 48 h post-infection, the cells were washed with PBS, trypsinized and fixed with 4% formaldehyde. The number of GFP-positive cells was determined using flow cytometry (BD FACSAria™). All experiments were done in triplicates and the mean ( ± standard deviation) of the percentage of EGFP-positive cells that resulted from infection with each of the mutant viruses was expressed relative to those obtained with the wild-type virus.

2.6. In vitro assembly of CA To study the formation of mature-like VLPs (cores) from wt and mutant M-PMV CA proteins, the N-terminal Met of the prepared Met–CA had to be removed to enable the formation of the β-hairpin at its N-terminus necessary for the virus core assembly and infectivity (Abdurahman et al., 2007; Fitzon et al., 2000; Gross et al., 1998; Obr 110

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2.10. ELISA quantification of CA protein

3.1. In vitro assembly of immature VLPs

To quantify the amount of CA protein in cultivation media used in the single-round infectivity assay, a sandwich enzyme-linked immuno sorbent assay (ELISA) was used. M-PMV capsid protein (Met–CA) in 0.01 M PBS containing 1% Triton X-100 was used for the construction of a calibration curve. All samples were analyzed in triplicates in a 96well plate. Rabbit anti-M-PMV CA polyclonal antibody (100 μl; 1:1000 dilution) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) was loaded into each well and immobilized during overnight incubation at 4 °C. The plate coated with captured antibody was washed with 0.01 M PBS containing 0.05% Tween-20 and 100 μl of either a CA-containing sample (i.e. the virus-containing medium (diluted 1:9 in 0.01 M PBS containing 1% Triton X-100)) or a standard curve construction sample (i.e. Met–CA) were added per well. After 1 h of incubation at 37 °C, the plate was washed with 0.01 M PBS containing 0.05% Tween-20 and incubated with 100 μl of rabbit anti-M-PMV CA antibody conjugated with horseradish peroxidase (HRP; 1:500 dilution in 0.05 M carbonatebicarbonate buffer (pH 9.6) for 90 min at 37 °C. After the washing step in 0.01 M PBS containing 0.05% Tween-20, the TMB substrate (3,3′,5,5′-tetramethylbenzidine, Sigma-Aldrich) for HRP was added and HRP was detected according to the manufacturer's instructions. The calibration curve was used to estimate the concentration of CA protein in samples based on their optical density reading.

To determine the effect of mutations of M-PMV CA cysteines on the assembly of immature VLPs, we firstly analyzed the ability of truncated mutant versions of M-PMV Gag (ΔProCANC) to form particles in bacteria. ΔProCANC was previously shown to mimic the immature M-PMV particle assembly, since the removal of the N-terminal proline of CA prevents the β-hairpin formation characteristic for the mature virus particle (Bharat et al., 2012; de Marco et al., 2010; Obr et al., 2014; Ulbrich et al., 2006; Wildová et al., 2008). The structures assembled from ΔProCANC mutant proteins in bacterial cells were assessed using TEM. In contrast to the wt ΔProCANC protein that formed tubular and spherical VLPs in E. coli, only the C213A mutant assembled into spherical particles, while no other cysteine mutant protein formed any particles, indicating the importance of cysteines for CA oligomerization and VLP assembly in a crowded cellular environment (data not shown). Our previous results showed that in vitro, the wt ΔProCANC M-PMV could form either spherical or tubular particles, depending on the assembly conditions and the type of added nucleic acid (Bharat et al., 2012; de Marco et al., 2010; Ulbrich et al., 2006). The spherical particles were efficiently formed under non-reducing conditions in the presence of MS2 RNA, unlike the tubular particles that were preferentially formed under reducing conditions in the presence of λDNA (Bharat et al., 2012; de Marco et al., 2010). To further assess the role of cysteines in the assembly process, we purified wt and ΔProCANC mutant proteins and used them in an in vitro assay of immature-like particle assembly under non-reducing conditions in the presence of MS2 RNA and under reducing conditions in the presence of λDNA (Bharat et al., 2012; Kuznetsov et al., 2007; Ulbrich et al., 2006). We found that unlike the wt and the other ΔProCANC mutants, the C213R mutant protein quantitatively precipitated during purification. Therefore, we were unable to prepare this mutant in sufficient amounts for in vitro studies. The precipitation of the C213R ΔProCANC mutant suggests that replacing this negatively charged M-PMV CA cysteine residue with a positively charged arginine residue negatively influences the structure of M-PMV CA, resulting in protein instability and precipitation. TEM analysis of the formed structures showed that similarly to the wt protein, the C82S, C82I, C193A, C193S and C213A ΔProCANC mutant proteins assembled in vitro under non-reducing conditions in the presence of MS2 RNA into spherical particles (Fig. 1A). However, under reducing conditions in the presence of λ DNA, the wt protein formed tubular VLPs and the aforementioned mutants formed a mixture of spherical and multi-layered particles (Fig. 1B). Also, the C213S, C82S/ C193S and C82S/C213S ΔProCANC mutant proteins efficiently assembled spherical particles in vitro. Their morphology somewhat differed based on their assembly conditions. Reducing conditions were more favorable for the formation of spherical VLPs without major structural defects, in contrast to non-reducing conditions that albeit supported the assembly of spherical particles from C213S, C82S/C193S and C82S/C213S ΔProCANC mutants, but with structural defects. The C193I ΔProCANC mutant protein assembled VLPs of spherical morphology only in the presence of reducing agent and λ DNA. The C193S/ C213S and C82S/C193S/C213S ΔProCANC mutant proteins did not assemble in vitro into any regular VLPs under any of the studied conditions; i.e. in the presence of MS2 RNA under non-reducing conditions and in the presence of λ DNA under reducing conditions (Fig. 1) but formed only filamentous multimers that have also been observed in C193I and C82S/C213S mutants (Fig. 1).

2.11. Transmission electron microscopy In vitro-assembled M-PMV VLPs or cores were deposited on a carbon-coated copper grid for 3–6 min. The grid was washed twice with deionized water and negatively stained with 4% sodium silico tungstate (pH 7.4) for 20 s. The excess stain was removed with filter paper and the samples were dried in air. For the ultra-thin section electron microscopic analysis of the assembly of wt/mutant M-PMV in HEK 293 T cells, the cells were prefixed 48 h post-transfection with freshly prepared 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 30 min and then scraped into a microtube. After washing with 0.1 M cacodylate buffer (pH 7.4), the cells were post-fixed with 1% osmium tetroxide for 1 h, dehydrated in an ethanol series (30%, 50%, 70%, 90%, 95% and 100% ethanol) and embedded in fresh AGAR 100 epoxy resin (Agar Scientific, UK). Ultrathin sections (~70 nm) of cells were cut with a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) using a diamond knife, collected on Parlodion®-coated microscopy grids and subsequently contrasted using saturated uranyl acetate and Reynold's lead citrate solutions. The samples were analyzed using a JEOL JEM-1010 transmission electron microscope (Jeol, Japan) operated at 80 kV, equipped with an SIS Megaview III CCD camera. The images were processed using the AnalySIS software suite (Olympus, Japan).

3. Results Unlike most retroviruses with two conserved cysteines in CA-CTD, M-PMV CA-NTD also contains a cysteine at position 82. To study the role of the cysteines present in M-PMV CA protein, we prepared a set of single, double, and a triple mutant where all cysteines (one in CA-NTD and two in CA-CTD) were mutated either to neutral alanine (single mutations C193A and C213A), isosteric serine (single mutations C82S, C193S and C213S, double mutations C82S/C193S, C82S/C213S and C193S/C213S, and triple mutation C82S/C193S/C213S) or to residues present at homologous positions in HIV-1 CA (C82I) or RSV CA (C193I, C213R) (see Fig. S1, Supplementary section). These mutations were introduced both into bacterial expression (pSIT ΔProCANC M-PMV, pTriExT Met–CA M-PMV) and M-PMV proviral (pSARM-EGFP) vectors.

3.2. In vitro assembly of mature VLPs Since the interaction interfaces of CA in the hexameric lattice are different in the immature and mature particles, we performed a mutagenesis study to investigate the role of cysteine residues in M-PMV CA in the assembly of mature M-PMV cores in vitro. To obtain mature VLPs, we removed the N-terminal methionine which blocks β-hairpin 111

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Fig. 1. TEM analysis of in vitro-assembled immature M-PMV particles negatively stained with 4% sodium silico tungstate, pH 7.4. Mutant/wt ΔProCANC particles were assembled in vitro in the presence of bacteriophage MS2 genomic RNA (10:1 (w/w) ratio of protein to nucleic acid) under non-reducing conditions (A) and bacteriophage λ genomic DNA (10:1 (w/w) ratio of protein to nucleic acid) under reducing conditions (60 mM DTT was included in the reaction mixture and 20 mM DTT in the assembly buffer) (B). Scale bars represent 200 nm.

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were similar to those of the wt and no aberrantly processed Gag products were detected (Fig. 3A and B). Moreover, mutations C193A, C193S, C213A and C213S did not interfere with viral release and maturation, since the CA protein was present in the cultivation media of transfected cells (Fig. 3C). Interestingly, no CA protein or non-processed viral polyproteins were detected in the culture medium (after 16-h chase) of cells expressing the C193S/C213S mutant, indicating that the mutation of both conserved cysteine residues to serine inhibited the MPMV life cycle at some stage of its late phase. This is consistent with the result of Western blot analysis of pelleted material from the culture medium of transfected cells (Fig. 3D), suggesting that the C193S/C213S mutation abolished the virus release. Furthermore, we calculated relative amounts of wt/mutant M-PMV particles released from the transfected cells defined as the amounts of CA proteins in cell culture medium (after 16-h chase) relative to the amounts of intracellular viral polyproteins (before chase). The results showed that the efficiency of release of C193A, C193S and C213A mutant particles was similar to that of the wt virus, while in the case of C213S mutant it was substantially decreased and reached approximately 25% of that of the wt, and in the case of C193S/C213S mutant it was under the detection limit (Fig. 3E). To analyze the importance of the C193 and C213 residues for the assembly of immature and mature M-PMV particles in infected cells, we performed a TEM analysis of ultra-thin sections of HEK 293 T cells transiently expressing the wt and C193A, C193S, C213A, C213S and C193S/C213S mutants of M-PMV. Similarly to the wt, all the single cysteine mutants formed spherical immature particles inside the cytoplasm of transfected cells (Fig. 4A), indicating that the single mutations of conserved cysteine residues in M-PMV CA do not abolish the immature particle assembly and have no influence either on particle morphology or the morphogenetic type of the virus. On the contrary, the double mutant C193S/C213S failed to assemble in HEK 293 T cells, despite the fact that its expression of Gag, Gag-Pro and Gag-Pro-Pol polyprotein was unaffected (Fig. 3A). As expected, the wt, C193 and C213 single-mutant viruses were efficiently released from infected cells. The mutant viral particles were indistinguishable from those of the wt and contained cores with mostly tubular morphology (Fig. 4B), as is natural for the wt virus (Coffin et al., 1997). No released particles were detected in the case of C193S/C213S mutant. Since the single mutation of either of the conserved cysteine residues in M-PMV CA did not abolish immature and mature particle assembly, we determined the infectivity of these virions by using a single-round infectivity assay. Except for the C213A mutant, whose relative infectivity reached approximately 20% of the wt, the other conserved cysteine mutants were almost non-infectious (Fig. 5).

formation at the N-terminus, which is typical for mature-like CA. This removal was sufficient to trigger the assembly of mature-like VLPs from M-PMV CA proteins, since the protein solutions after the cleavage turned opalescent and mature-like particles were detected by TEM. The efficiency of the assembly process was additionally enhanced by the addition of PEG 3000, which acts as a crowding agent, thus mimicking the dense environment inside a virion and helping to greatly reduce the critical protein concentration needed for CA assembly in vitro, as was also reported for HIV-1 (del Álamo et al., 2005). All multiple-cysteine mutants, i.e. the C82S/C193S, C82S/C213S, C193S/C213S and C82S/ C193S/C213S M-PMV Met–CA proteins, were poorly soluble, even though they were expressed in N-terminal fusion with yeast SUMO (small ubiquitin-related modifier) protein Smt3, which should increase the protein expression level and its solubility in bacterial cells (Lee et al., 2008; Malakhov et al., 2004). Although the Smt3 fusion partially increased their solubility, these proteins quantitatively precipitated immediately after the His–Smt3 tag cleavage, which prevented the study of their in vitro assembly. In contrast, the wt and C82S, C193S and C213S M-PMV Met–CA mutant proteins were soluble, monomeric and stable in low salt buffer (50 mM Tris, 100 mM NaCl, 5 mM TCEP, pH 8.0) even at high protein concentrations (10–30 mg/ml). TEM analysis showed that the wt as well as the C82S, C193S and C213S M-PMV CA mutant proteins assembled in vitro into mature-like VLPs of various morphologies (Fig. 2), mostly mimicking the tubular or conical core that is present in infectious M-PMV virions. However, in the case of mutant CA proteins, these structures were often aberrant, and mature-like VLPs assembled from the C213S mutant protein even exhibited the presence of aggregated protein (Fig. 2). The obtained wt and mutant VLPs were very fragile (unstable), since even the small dilutions of solutions containing VLPs led to their disintegration. This is in agreement with the previously mentioned study on HIV-1 (del Álamo et al., 2005). Thus, proper sample preparation and fixation with glutaraldehyde for subsequent TEM analysis was crucial for visualization of the particles. 3.3. The effect of cysteine mutations on M-PMV life cycle and infectivity The single mutations of cysteines in M-PMV CA had only a minor effect on the assembly of both immature and mature VLPs in vitro. This was the case of mutations of the conserved cysteines C193 and C213; whereas the double mutation of these residues inhibited the assembly completely. Thus we further investigated the role of mutations of the conserved cysteines C193 and C213 that are more likely to be involved in the formation of disulfide bridges. For this purpose we introduced a series of cysteine to serine/alanine mutations of CA into the M-PMV proviral pSARM-EGFP vector and performed a pulse–chase experiment in transiently transfected HEK 293 T cells. The results showed that none of the studied mutations of conserved cysteine residues (C193A, C193S, C213A, C213S and C193S/C213S) in M-PMV CA significantly affected the expression or processing of viral structural polyproteins. The intracellular levels of Gag, Gag-Pro and Gag-Pro-Pol of mutant M-PMV

4. Discussion A pair of highly conserved cysteines that can form an intramolecular disulfide bond is present in the majority of retroviral Gag polyprotein CA-CTDs (Jin et al., 1999; Nath and Peterson, 2001; Worthylake et al.,

Fig. 2. TEM analysis of in vitro-assembled mature wt/mutant M-PMV CA particles negatively stained with 4% sodium silico tungstate, pH 7.4. M-PMV CA particles were formed from purified Met-CA protein after the removal of its N-terminal methionine and the addition of 250 mg/ml PEG 3000 in a 1:1 (v/v) ratio, serving as the crowding agent. Scale bars represent 200 nm. 113

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Fig. 3. Synthesis, release and processing of wt/mutant M-PMV. M-PMV CArelated proteins were immunoprecipitated with a rabbit anti-M-PMV CA antibody from cell lysates and supernatants from metabolically labeled HEK 293 T cells transiently expressing wt/mutant M-PMV. Pull-down proteins were separated by SDS-PAGE and analyzed by PharosFX™ Plus Molecular Imager. (A) Intracellular M-PMV polyproteins Gag, Gag-Pro and Gag-Pro-Pol immunoprecipitated from the cell lysates after 30-min pulse and (B) after 16-h chase. (C) CA-related proteins of released M-PMV particles immunoprecipitated from the cell supernatant after 16-h chase. (D) Western blot analysis with rabbit anti-M-PMV CA antibody of wt/mutant M-PMV particles collected by ultracentrifugation through 20% sucrose cushion of cell supernatant 72 h posttransfection. To normalize for producer cell number, we measured the levels of GAPDH in cell lysates by Western blot with a rabbit anti-GAPDH antibody. (E) Quantification of wt/mutant M-PMV particle release from HEK 293 T cells. Band intensities of intracellular [35S]-pulse-labeled Gag, Gag-Pro and Gag-ProPol and released CA were calculated by densitometry analysis. The amount of CA released into the cell supernatant was normalized to levels of intracellular proteins for each analyzed sample. Due to the presence of a single band of CA in each line, only cropped images are shown in panels C and D.

1999), except the alpharetroviruses (e.g. RSV) and spumaretroviruses, where these cysteines are absent. To study their role and importance in M-PMV, namely in immature particle assembly, retroviral core formation and virus infectivity, we prepared a set of M-PMV CA cysteine mutants. The effects of all these mutations were studied in both in vitro and in vivo systems. M-PMV Gag and its mutant carrying the fusion CA-NC protein with the deletion of an N-terminal amino acid, proline, i.e. ΔProCANC, can

form in vitro virus-like particles (VLPs) with the arrangement of the immature lattice (Bharat et al., 2012). The oligomerization of CA may lead to two distinct morphologies of in vitro-assembled immature-like particles: spherical under non-reducing conditions and tubular under reducing conditions (Bharat et al., 2012; Füzik et al., 2016; Ulbrich et al., 2006). These results prompted us to evaluate whether the disulfide bonds present in M-PMV ΔProCANC affect the assembly, morphology and stability of immature M-PMV particles. Except for the C213R mutant protein that precipitated during the purification process, all other single cysteine mutant proteins assembled in vitro into spherical or multi-layered particles, depending on the assembly reaction mixture composition, indicating the role of cysteines in the morphology of formed particles. The assembly of the double mutants was severely impaired, as they formed a high proportion of disrupted and unstable particles. These results indicate that the presence of at least one of the conserved cysteine residues in the M-PMV CA protein is substantially important for VLP assembly in vitro. This observation agrees with previously published mutagenesis studies of HIV-1 and FIV, where the mutations of one of conserved residues lead to assembly disruption or protein thermal instability (McDermott et al., 1996; Nath and Peterson, 2001). If both conserved cysteines are mutated, it either highly changes the protein native structure or leads to disulfide-bond disruption, resulting in the destabilization of formed VLPs. None of the in vivo-studied mutations affected the M-PMV polyprotein expression in transfected HEK 293 T cells. In agreement with the in vitro data, all the M-PMV single cysteine mutants assembled intracytoplasmic immature particles, which budded through the plasma membrane. The only single mutation with altered virus production was the C213S mutation, which led to a significant decrease in relative particle release compared to wt M-PMV. A similar importance of conserved cysteine residues was also observed in HIV-1, where the C198S mutation did not affect the assembly and particle release of HIV-1, whereas the C218S mutation virtually eliminated the particle assembly (McDermott et al., 1996). In agreement with in vitro assays, the C193S/ C213S mutation led to the complete inhibition of immature particle assembly and release from infected cells. The single cysteine mutant particles released from the cells were mature with visible core structures indistinguishable from the wt virus. Surprisingly, none of the M-PMV single cysteine mutant viruses were infectious. Therefore, the stability of the formed mature cores was questionable. The in vitro CA assembly assay showed the presence of protein aggregates in the case of C213S mutant CA protein, which could indicate a lower efficiency of the core assembly of this mutant compared to the wt and C193S, suggesting the importance of C213, possibly by stabilizing the protein. In contrast to HIV-1, the M-PMV native cores were highly unstable, which prevented a stability comparison of wt and

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Fig. 4. TEM analysis of ultra-thin sections of HEK 293 T cells expressing wt/mutant M-PMV proteins. The HEK 293 T cells were transiently co-transfected with wt/ mutant M-PMV-EGFP- and Env-expression vectors. At 48 h post transfection, the cells were prefixed with 3% glutaraldehyde and post-fixed with 1% osmium tetroxide, dehydrated in an ethanol series and embedded in epoxy resin. Ultra-thin sections were contrasted using saturated uranyl acetate and Reynold's lead citrate and analyzed using a JEM-1010 transmission electron microscope. (A) Immature M-PMV particles assembled inside cells. (B) Mature virions released from cells. Scale bars represent 200 nm.

suggest that the cysteine residues present in M-PMV CA stabilize the protein in vitro. Taken together, the presence of both intact conserved cysteine residues in M-PMV CA is necessary for the virus infectivity. Although mutations of either of the conserved cysteine residues in M-PMV CA do not inhibit the assembly of immature viral particles and influence virus core formation, the double mutation of both conserved cysteine residues abrogated the particle

mutant cores. The replacement of both conserved cysteine residues with isosteric serines resulted in the complete inhibition of CA assembly in vitro, indicating the important role of conserved cysteine residues in CACTD in protein stability and retroviral assembly. This is consistent with similar findings that highly conserved, disulfide-bonded cysteines, but not the disulfide bond itself, are important for the conformational stability of the HIV-1 CA-CTD monomer (Mateu, 2002). Our results also 115

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Fig. 5. Relative infectivity of wt/mutant M-PMV determined by single-round infectivity assay. HEK 293 T cells were transiently co-transfected with wt/mutant M-PMV-EGFP- and Env-expression vectors. EGFP expression was detected by flow cytometry in HEK 293 T cells 48 h post infection with CA-normalized, single-round infection M-PMV-EGFP viruses. The experiments were done in triplicates and the mean ( ± standard deviation) of the percentage of EGFPpositive cells that resulted from infection with each of the mutant viruses was expressed relative to those obtained with the wild-type virus.

assembly. We may speculate that the presence of both intact conserved cysteine residues is necessary for maintaining the proper CA-CTD structure and subsequent Gag oligomerization. This correlates with HIV-1, where these conserved cysteine residues are also present in the last two α-helices (i.e. H10 and H11) of HIV-1 CA-CTD and might influence proper H10 and H11 orientation critical for inter-molecular CA–CA interaction (Byeon et al., 2009). Thus, the cysteines present in the capsid protein, a major structural part of both the mature and immature particle, are highly likely to influence the stability of mature cores and thus influence the infectivity of the mature particles. Acknowledgments This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic [grant number LTAUSA17061]; and the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA [grant number SC1GM115240]. Declaration of conflicts of interest None. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2018.06.001. References Abdurahman, S., Youssefi, M., Höglund, S., Vahlne, A., 2007. Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity. Retrovirology 4, 69. http://dx.doi. org/10.1186/1742-4690-4-69. Alfadhli, A., Barklis, R.L., Barklis, E., 2009. HIV-1 matrix organizes as a hexamer of trimers on membranes containing phosphatidylinositol-(4,5)-bisphosphate. Virology 387, 466–472. http://dx.doi.org/10.1016/j.virol.2009.02.048. Bharat, T.A.M., Davey, N.E., Ulbrich, P., Riches, J.D., de Marco, A., Rumlova, M., Sachse, C., Ruml, T., Briggs, J. A.G., 2012. Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy. Nature 487, 385–389. http://dx.doi.org/10. 1038/nature11169. Bohmová, K., Hadravová, R., Štokrová, J., Tůma, R., Ruml, T., Pichová, I., Rumlová, M., 2010. Effect of dimerizing domains and basic residues on in vitro and in vivo assembly of Mason-Pfizer monkey virus and human immunodeficiency virus. J. Virol. 84, 1977–1988. http://dx.doi.org/10.1128/JVI.02022-09.

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