The Assembly Domain of the Small Capsid Protein of Kaposi’s Sarcoma-Associated Herpesvirus Dale Kreitler,a Christopher M. Capuano,a Brandon W. Henson,a Erin N. Pryce,b Daniel Anacker,a J. Michael McCaffery,b and Prashant J. Desaia Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,a and Integrated Imaging Center,b Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA
Self-assembly of Kaposi’s sarcoma-associated herpesvirus capsids occurs when six proteins are coexpressed in insect cells using recombinant baculoviruses; however, if the small capsid protein (SCP) is omitted from the coinfection, assembly does not occur. Herein we delineate and identify precisely the assembly domain and the residues of SCP required for assembly. Hence, six residues, R14, D18, V25, R46, G66, and R70 in the assembly domain, when changed to alanine, completely abolish or reduce capsid assembly.
T
he Kaposi’s sarcoma-associated herpesvirus (KSHV) capsid shell is made up of four proteins: the major capsid protein (MCP) encoded by open reading frame 25 (ORF25), triplex proteins 1 and 2 encoded by ORF62 and -26, respectively, and the small capsid protein (SCP) encoded by ORF65. The internal scaffold proteins (ORF17.5) are important for icosahedral capsid assembly and the serine protease (ORF17) for maturation of the capsid during genome packaging (1, 2, 6, 15, 17). Self-assembly of capsids using recombinant baculoviruses for protein expression has been demonstrated for herpes simplex virus type 1 (HSV-1) (10, 11, 18, 19) and subsequently for KSHV and Epstein-Barr virus (EBV) (5, 13). The assembly pathway of gammaherpesviruses (4, 9, 20, 21) was similar to that of HSV-1 with one exception: the SCP was essential for self-assembly (5, 13). This was confirmed by mutation of ORF65 in a KSHV bacterial artificial chromosome (BAC) clone in infected cells (16). The goal of this study was to use self-
assembly of KSHV capsids in insect cells in conjunction with baculovirus expression systems to discover the assembly domain of the SCP and the important amino acids within this domain. To do this, we used PCR methods to make N-terminal and C-terminal truncations of ORF65 using pFastBac1-ORF65 as a template (13), and we transferred these into the baculovirus ex-
Received 8 June 2012 Accepted 6 August 2012 Published ahead of print 22 August 2012 Address correspondence to Prashant Desai,
[email protected]. Dale Kreitler, Christopher M. Capuano, Brandon W. Henson, and Erin N. Pryce equally contributed to this study. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.01430-12
FIG 1 Assembly domain of the KSHV SCP. Polypeptide truncation mutants were expressed in Sf21 cells to determine the assembly domain of ORF65. (A) Amino acid sequence of ORF65 and representation of the N- and C-terminal truncation mutants made for this experiment. The ORF65 gene encodes a 170-amino-acid polypeptide. Under CAPSID are the results of the assembly experiments. (B) Western blot analysis of SF21 cells infected with the recombinant baculoviruses expressing the different polypeptide truncation mutants. The protein standards are shown in the first lane, and the membrane was probed with anti-HA antibody (Roche clone 3F10) using previously described methods (3). The 65-amino-acid polypeptide was not detected using the NuPage (Invitrogen) gel system, and the 87-amino-acid polypeptide resolved into multiple protein species, most likely because of posttranslational modifications. (C) Negative-stained images of KSHV capsids harvested from sucrose gradients following sedimentation of infected cell lysates performed using methods described by Perkins et al. (13). Scale bars are all 100 nm except for the second 170 image, for which the bar is 200 nm. A cosedimenting baculovirus particle can also be seen in the 125 panel.
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KSHV ORF65 Assembly Domain and Functional Properties
FIG 2 Amino acids R14, D18, V25, R46, G66, and R70 are important for assembly of KSHV capsids. (A) An alignment of the N-terminal 89 amino acids of the gammaherpesvirus SCPs is shown (ClustalW algorithm in MacVector). (B) ORF65 N-terminal 86-amino-acid sequence; all the residues underlined and in bold were changed to alanine. (C) Western blot analysis of infected Sf21 cells (harvested 48 h postinfection) showing stable expression of the ORF65 mutants except for Y58A. Protein standards are shown in the first lane, and the membrane was probed with anti-HA antibody (WT, wild-type; MI, mock-infected). (D) Quantitation of the peak fractions from sucrose gradients. Radiolabeled lysates were sedimented through sucrose gradients and fractionated, and the 35S counts per minute were determined in the peak fraction. The data derived from 2 or 3 independent experiments were plotted and the numbers derived by calculating the percentage of counts relative to the wild-type samples in each experiment. (E) Biochemical analysis of sucrose gradient fractions. Radiolabeled lysates were sedimented through sucrose gradients, and fractions 7, 8, and 9 (F7, F8, and F9, respectively) were examined by SDS-PAGE (7). Capsids normally band in fraction 8, as was observed for the wild-type ORF65 protein. For V25A, the radioactivity detected in the fraction was much lower and there was no evident increase in radioactivity in F8. The mobilities of the capsid proteins in the gel are indicated on the right of the gel. Protein standards are shown in lane M and are 97.4, 66, 46, 30, and 14.3 kDa.
pression vector pFastBac1 (Invitrogen) in order to test them for assembly. The forward primer specified a SpeI site and an inframe N-terminal influenza hemagglutinin (HA) epitope, and the reverse primer specified a HindIII site with the corresponding truncation (Fig. 1A). Spodoptera frugiperda (Sf9 and Sf21) cells were used for these experiments and propagated as described by Okoye et al. (12). Recombinant baculovirus generation methods are described by Okoye et al. (12) and Perkins et al. (13). Expression of the truncated polypeptides was confirmed by Western blots using anti-HA antibody (Fig. 1B). To facilitate our self-assembly experiments we also made dual-expression baculovirus recombinants. Thus, we cloned ORF25 and ORF17.5 into pFastBac Dual (Invitrogen) and we did the same for ORF62 and ORF26 (C. M. Capuano, D. Kreitler, B. H. Henson, E. N. Pryce, J. M. McCaffery, and P.J. Desai, unpublished data). We then coinfected Sf21 cells with FBD-ORF17.5/25, FBD-ORF26/62, FBORF17 (13), and the baculovirus expressing wild-type or mutant ORF65 polypeptides. The coinfected cells were harvested and the lysates examined for capsids by sucrose gradient sedimentation
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and electron microscopy (EM) of negative-stained material from the gradients (Fig. 1C). Visually, mutants expressing truncations at amino acids 86 and 125 gave strong light-scattering bands in sucrose gradients and closed icosahedral capsids when examined by EM. Mutants expressing truncations at amino acids 65 and 75 and the N-terminal truncation 87 did not yield capsids as judged by either method (data not shown). The 100-amino-acid mutant did not yield any capsids in several experiments. Because truncation at amino acid 86 supports assembly, we concluded that truncation at 100 amino acids, although stably expressed, may be misfolded. Thus, the assembly domain as shown for EBV SCP (5) resides in the N-terminal half of ORF65 polypeptide. We next aligned the amino acid sequences of the gammaherpesvirus SCP, specifically focusing on the N-terminal 86 amino acids, to discover conserved residues within the assembly domain that could be targeted for mutagenesis. A major mutagenesis effort was made on several residues that were changed to alanine (Fig. 2A and B). The mutants were made in pFastBac1 containing the ORF65 gene and an HA epitope at the C terminus. To make
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TABLE 1 Summary of the self-assembly assay for the ORF65 mutants ORF65 mutant
Capsid assemblya
K5A D8A Q12A E13A R14A L15A D16A H17A D18A Y19A L24A V25A R27A L31A Q33A N35A R46A Y48A L49A V50A F51A L52A I53A Y57A Y58A E59A Y61A R63A R64A M65A G66A R70A R71A D79A R83A
⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹
a
⫹, capsid assembly; ⫺, no capsid assembly.
this plasmid, ORF65 was amplified and cloned as a SpeI-HindIII fragment; the reverse primer specified the HindIII site and the HA epitope sequence (pFB1-ORF65CHA). An internal EcoRI recognition site in ORF65 was inactivated by silent site mutagenesis using QuikChange (Stratagene), and this plasmid served as the template for mutagenesis (pFB1-ORF65CHA-⌬EcoRI). ORF65 was also cloned into pFB1-CHA (5) as a BamHI-SpeI fragment (pFB1-ORF65CHABS). Mutants were made by QuikChange (7) or cassette PCR methods using Pfu Ultra (Stratagene) or Phusion polymerase (Finnzyme-NEB). The cloned wild-type and mutant genes were sequenced to check for authentic amplification. Mutants K5A and D8A were cloned as EcoRI-SpeI fragments into pFB1-CHA. The forward EcoRI primer contained the mutation. Mutants Y57A, Y58A, E59A, and Y61A were PCR amplified as BamHI-EcoRI fragments. The reverse primer contained the mutation and an EcoRI site. The resulting DNA fragment was cloned into a pFB1-ORF65CHABS vector. All the mutants were tested for self-assembly using coinfections of Sf21 cells (Table 1). Visually, many mutants gave strong light-scattering bands in sucrose gradients; this was confirmed by EM of material from the gradients (data not shown). For mutants R14A, D18A, V25A, R46A, Y58A, G66A, and R70A we did not detect a light-scattering band in the sucrose gradients and when
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material at the position where KSHV B capsid sediment was examined by EM, assembled capsids were not evident, indicating the loss of ORF65 assembly function. The stable accumulation of ORF65 expressed by these mutants was examined in insect cells using Western blots (Fig. 2C). All mutants except Y58A expressed the ORF65 polypeptide. To demonstrate the assembly defect quantitatively, infected Sf21 cells (1 ⫻ 107) were labeled with 250 Ci of 35S-Express (Perkin Elmer) in unsupplemented Grace’s insect cell medium from 42 to 72 h postinfection and then processed for sedimentation. Radioactivity in the peak fraction containing capsids was determined (Fig. 2D), and the data show that radioactivity in this fraction for all mutant ORF65 gradients was 10-fold lower than in wild-type ORF65. The peak fractions were also examined for the presence of cosedimenting capsid proteins by SDS-PAGE (Fig. 2E). In the presence of wild-type ORF65, radioactivity corresponding to the MCP, scaffold, and triplex proteins was evident in fraction 8. When mutant V25A polypeptide was present, radioactivity corresponding to the shell and scaffold proteins was detected but the signal was decreased and the same in all fractions. Furthermore, radioactivity corresponding to ORF65 was not evident. Because R14A, D18A, and G66A mutants gave weak bands in sucrose gradients when the number of cells analyzed was increased, we examined all mutant infected cells by transmission EM methods (Fig. 3). In the nuclei of infected cells, numerous cylindrical structures/tubules of varied lengths were observed (white arrow). Open capsid shells were also evident but, on close examination, are different from these shells and have the same repetitive indentation present in the long cylinders (black arrowheads, R46A right panel and V25A upper panel). Small circular structures of uniform size and a central core were also observed (white arrowheads, R70A panel and V25A lower panel), these could also be cross sections of the tubular structures. It is likely that all these structures are composed of the scaffold protein, based on data that show similar structures for the VZV scaffold protein (14) and for a mutant HCMV scaffold protein that has a weakened interaction with MCP (8). We did not see these structures when KSHV capsid assembly did not occur, but ORF17.5 was present (13). None of these structures were evident in the sucrose gradient fractions examined for the mutants. This could be because they sediment to the bottom in the rate velocity sedimentation experiments or they are not stable in sucrose gradients. In cells infected with the G66A and D18A mutants we also observed regular icosahedral capsid structures (black arrows). How does ORF65 fit into this phenotype? Our hypothesis is that the mutant ORF65 polypeptides can still interact with MCP and they interfere with the MCP-scaffold protein interaction during shell accretion that causes the scaffold protein to disengage from the interaction with the outer shell and begin or continue an irreversible reaction that leads to the accumulation of elaborate scaffold structures. In summary, the gammaherpesvirus SCP is an important mediator of stable capsid shell assembly and thus a new antiviral target. In this study, we show that the assembly domain of ORF65 resides in the N-terminal 86 residues. We made 35 alanine substitution mutations in the assembly domainof ORF65, and 6 abolish assembly. Mutants V25A, R46A, and R70A completely abrogate assembly, whereas mutants R14A, D18A, and G66A could support limited icosahedral capsid assembly. Thus, new sites of this pro-
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FIG 3 Ultrastructural analysis of coinfected Sf21 cells. Cells were coinfected with viruses FBDORF17.5/25, FBDORF26/62, FBORF17, and the virus expressing the wild-type or mutant ORF65 polypeptide. Cells were processed for TEM 68 h after infection, according to procedures described by Perkins et al. (13). Scale bars: R46A (left), 1,000 nm; R46A (right) and V25A (top), 400 nm; G66A (middle) and D18A, 200 nm; G66A (left), R70A, WT, and V25A (lower), 100 nm.
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tein have been discovered that could form the basis for discovery of a small-molecule inhibitor.
10.
ACKNOWLEDGMENTS Funding for this research was provided by PHS grants from the NIH AI061382 and ARRA funding in support of summer students AI061382-S4 (P.J.D.). Additional funding for shared equipment was provided by NIH-NCRR, 1S10RR023454-01 (shared instrumentation grant for the Tecnai 12 G2 Spirit TEM), and NIH-NCI 1U54CA143868-01 (Physical Sciences of Cancer grant) (J.M.M.). We thank Helen Benton for help with cloning HA-tagged ORF65.
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