Phosphorylation of Marburg Virus NP Region II Modulates Viral RNA Synthesis

Abstract

Phosphorylation of the Marburg virus nucleoprotein NP is distributed over 7 regions (I—VII) in its C-terminus. The exact localization of phosphorylated amino acids and function of NP phosphorylation are unknown. Here, we show that the major phosphate acceptor sites in NP region II are serine 446 and serines 453–455; the latter are located in a cluster of 6 serine residues (aa 450–455). The function of phosphorylation in region II was tested using an infectious virus-like particle assay. Phosphorylation influenced reporter gene activity that reflects viral transcription and replication. An NP mutant mimicking 3 phosphorylated serine residues at position 453–455 supported reporter gene activity better than wild-type NP. Negative charges at positions 450–452 and when the serine cluster was completely substituted by alanine inhibited reporter gene activity significantly. These data support the idea that phosphorylation of NP region II modulates viral RNA synthesis in transcription and/or replication.

Marburg virus (MARV) and the closely related Ebola virus together make up the family of Filoviridae, which is classified in the order Mononegavirales. MARV causes a fulminant hemorrhagic fever in humans and nonhuman primates with high fatality rates [1]. Neither a vaccine nor a curative treatment for MARV infection of humans is available.

The MARV particle is composed of 7 structural proteins. Four of them, NP, VP35, VP30, and L, form the nucleocapsid complex of MARV, which surrounds the viral genome [2]. NP, the major nucleocapsid protein, self-assembles into tubular nucleocapsid-like structures, which are mainly found in large intracellular inclusions [3–5]. NP interacts with VP35, which, in turn, interacts with the RNA-dependent RNA polymerase L [2, 6]. The complex of VP35 and L acts as the active RNA-dependent RNA polymerase, with VP35 serving as polymerase cofactor [7]. Additionally, a trimeric complex was observed consisting of NP, VP35, and L with VP35 connecting L and NP [2]. Three of the 4 nucleocapsid proteins, NP, VP35, and L, are essential for transcription and replication of the viral RNA [8]. The fourth nucleocapsid protein, VP30, plays an important role in viral transcription in the closely related Ebola virus [7, 9–11]. For MARV, the role of VP30 is not completely understood at this time. Although a minigenome-based transcription/replication system did not indicate a requirement for VP30 in transcription [8], RNAi-based downregulation of VP30 expression in MARV-infected cells resulted in decreased levels of all other viral proteins. In addition, VP30 had supportive effects on viral replication and transcription in a recently published infectious virus like particle assay [12].

From the 7 MARV proteins, NP, VP30, and GP are phosphorylated [3, 13, 14]. Although it is known that the nucleoprotein of MARV is highly phosphorylated, the function of this posttranslational modification during the viral life cycle is still unknown [3]. In MARV infected cells, 2 forms of NP are detectable (92 and 94 kDa), from which only the 94 kDa form is incorporated into viral particles. The 94 kDa NP can be converted into the 92 kDa form by dephosphorylation, and it was suggested that phosphorylation triggers the recruitment of NP into MARV particles [3]. Although most phosphorylated nucleoproteins of Mononegavirales have only a few phosphorylation sites, MARV NP has 7 regions that are phosphorylated, and only a few of the phosphorylated amino acids have been mapped [15]. The phosphorylated serine and threonine residues are distributed over the variable C- terminus, whereas the conserved N-terminus of NP up to amino acid (aa) 389 is not phosphorylated [15]. Previously, 7 phosphorylated regions have been determined in the C-terminus of NP; one of which, region II, contains a characteristic cluster of 6 consecutive serine residues and several negatively charged amino acids, which together make this region a prime candidate to be phosphorylated by cellular protein kinases. Here, we show that major phosphate acceptor sites in region II are serines 446 and 453–455. Mutation of these serine residues to either aspartate or alanine influenced the function of NP in transcription/replication. NP activity was prominently down-regulated in the early stages of infection, when all serines of the cluster were replaced by aspartate. Negative effects were also observed when the last 3 serines of the cluster were replaced by alanine.

METHODS

Cell Culture and Virus

HUH7, HEK293, and HeLa cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 5 mM of glutamine, and antibiotics at 37°C and 5% CO₂. The vaccinia virus MVA-T7 was grown as described elsewhere [8]

Construction of NP Mutants

NP^regII (aa 439 to 475 of MARV NP) (EMBL nucleotide sequence database accession no. Z12132) was amplified by polymerase chain reaction and subcloned into pGEM-NP, which had been pretreated with the restriction endonuclease PstI. The resulting construct encoded the nonphosphorylated N-terminus of NP fused to region II (pGEM-NP^regII) [15]. Substitutions of single amino acids in pGEM-NP^regII (pGEM-NP^regII458A, pGEM-NP^regII458/475A, NP^regII446A, NP^regIIAS, and NP^regIIAA) were obtained by site-directed mutagenesis using pGEM-NP^regII as template (Stratagene; Clontech). All clones encoding full-length NP containing mutations in the serine cluster of region II (NP_AA, NP_DD, NP_AD, and NP_DA) were generated by using pT-NP as template and site -directed mutagenesis [8]. Resulting NP-encoding DNA was subcloned into the expression vector pCAGGS [12]. Full-length NP constructs contained an N-terminal HA-tag that did not affect NP function or localization (authors’ unpublished data). Detailed cloning strategy and primer sequences are available on request. All constructs have been verified by sequencing.

MVA-T7 Expression System

Expression and metabolic labeling of NP in HeLa cells (70% confluent) were performed as described by Lötfering et al [15], except that MVA-T7 was used instead of the vaccinia virus vTF7/3.

Indirect Immunofluorescence Analysis (IFA)

HUH7 cells were transfected with 500 ng of pCAGGS NP or NP mutants, VP35 or VP30, respectively, using TransIT (Mirrus). IFA was performed as described elsewhere [16]. NP was stained using guinea pig anti-NP (dilution, 1:50) and TexasRed-labeled goat anti-guinea pig or a monoclonal anti-NP antibody (dilution, 1:50), followed by a rhodamine-labeled goat anti-mouse antibody. VP35 was stained using a guinea pig anti-VP35 antiserum (dilution, 1:50) and an fluorescein isothiocyanate (FITC)-labeled goat anti-guinea pig antibody. VP30 was detected using a monoclonal anti-VP30 mouse antibody (dilution, 1:100) and a secondary FITC-labeled goat anti-mouse antibody. All secondary antibodies were diluted to 1:100. Nuclei of cells were stained with DAPI (dilution, 1:10000).

Metabolic Labeling of NP and Immunoprecipitation

Labeling of NP with [³⁵S] methionine or [³²P_i] and subsequent immunoprecipitation of radio-labeled proteins was performed as described by Modrof et al [13].

Phosphoamino Acid Analysis (2-TLC)

Phosphoamino acid content was determined as described elsewhere [15]. Quantification was done with Raytest Basreader and TINA 2.1 software.

In Vitro Kinase Assay for Protein Kinase CKII

HeLa cells were infected with MVA-T7 and transfected with plasmids encoding for NP or NP mutants. At 16 hours p.i., cells were lysed in CoIP buffer [17] (20 mM of Tris-HCL; pH, 7.6; 100 mM of NaCl, 5 mM of EDTA, 1% [w/v] NP40, 0.4% [w/v] deoxycholate) in presence of 100 μM of sodium orthovanadate, 4 mg/mL soybean trypsin inhibitor, 10 μg/mL leupeptin, 2.8 mg/mL aprotinin and 500 μg/mL pepstatin on ice for 30 minutes. Lysates were cleared by centrifugation at 13000 g for 15 minutes, and the supernatants were used for NP-specific immunoprecipitation. Immobilized NP or mutants of NP were dephosphorylated at 37°C for 45 minutes using 20 U calf intestinal phosphatase (Roche) in 50 μL of CIP buffer (Roche). Dephosphorylated immobilized proteins were washed 3 times with ice-cold PBS and used for in vitro phosphorylation with recombinant protein kinase CKII (Roche) as described elsewhere [18].

MARV-Specific Infectious Virus-Like Particle (iVLP) Assay

Functional analysis of mutated full-length NP was performed using a MARV-specific iVLP assay, as described by Wenigenrath et al, [12]. Producer cells were lysed at 72 hours p.t,. and Renilla reporter activity was measured using the Dual Luciferase Assay (Promega). Supernatants of cells containing infectious virus like particles were collected and used for infection of indicator cells that had been previously transfected with plasmids encoding the nucleocapsid proteins NP, VP35, VP30, and L. When NP had been replaced by mutants of NP in the producer cells, the same mutants were used to pretransfect the indicator cells. Indicator cells were lysed at 60 hours p.i., and reporter acitivty was determined. Results gained with wild-type NP were set to 100%. An aliquot of iVLPs was used for incorporation studies using protease protection assay, as described elsewhere [12].

RESULTS AND DISCUSSION

Identification and Localization of Phosphate Acceptor Sites in NP Region II

To determine the localization of phosphorylated amino acids in region II, the nonphosphorylated N-terminus of NP was fused to the phosphorylated region II (NP^regII) (Figure 1). NP^regII was expressed in HeLa cells in the presence of [³²P_i] or [³⁵S]methionine and subsequently immunoprecipitated using NP-specific antibodies and analyzed. NP^regII was phosphorylated, supporting previously published results (Figure 1) [15]. Phosphoamino acid analysis of NP^regII showed that mainly serine residues were phosphorylated, and a minor signal of phosphothreonine was detected (Figure 1C). Because several protein kinase CKII recognition sites were present in region II (S/TxxD/E), it was tested whether this kinase contributed to phosphorylation of region II. To this end, aspartate 458 and aspartate 475, which are essential residues of the conserved protein kinase CKII recognition sites (₄₅₅SFVD₄₅₈ and ₄₇₂TLDD₄₇₅ [Figure 2A]), were substituted by alanine and the phosphorylation state of the resulting constructs, NP^regII458A and NP^regII458/475A, was analyzed. Neither NP mutant showed a significantly weaker phosphorylation signal than NP^regII (Figure 2D, lanes 3 and 6). To confirm these results, we investigated whether NP^regII could be phosphorylated in vitro by protein kinase CKII (Figure 2C). In contrast to the full-length NP, which is readily phosphorylated by protein kinase CKII, NP^regII is not (Figure 2D, lanes 3 and 6). This data indicated that 2 additional protein CKII recognitions sites outside region II located in other previously-determined phosphorylated regions of NP (S549 and T417) might serve as phosphate acceptor sites for protein kinase CKII.

Figure 1.

Phosphorylation state analysis of region II of NP.A, Schematic presentation of NP and NP^regII. NP^regII is composed of the nonphosphorylated N-terminus of NP (aa 1–389) fused to region II of NP (aa 439–475). The amino acid sequence is shown in 1 letter code. B, Metabolic labeling of NP and NP^regII. HeLa cells were infected with MVA-T7 and transfected at 1 h p.i. with plasmids encoding NP and NP^regII. Cells were radiolabeled with [³²P_i] and [³⁵S]methonine. Immunoprecipitation of NP was performed using a polyclonal rabbit antiserum against Marburg virus nucleocapsid proteins (αNC; dilution, 1:500). The radiolabeled proteins, were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and detected by bioimage analyzer BAS-1000. Lane 3 shows MVA-T7 infected cell lysate, and lane 4 shows mock-infected cell lysate C. Phosphoamino acid analysis of NP^regII. HeLa cells were treated as described in B. The radio-labeled proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to Polyvinylidendifluorid membrane and phosphoamino acid analysis (2D-TLC) was performed. Radioactive phosphoamino acids were detected by bioimage analyzer BAS-1000.

Figure 2.

Functionality of protein kinase CKII acceptor sites in region II. A, Schematic presentation of NP^regII substitution mutants NP^regII 458A and NP^regII 458/475A. B, Metabolic labeling of NP. HeLa cells were infected with MVA-T7, transfected, and radio-labeled as described in Figure 1. NP and mutants of NP were immunoprecipitated, and radioactive signals were quantified (n = 4). The phophosignals were normalized by [³⁵S]methionine signals. Phosphorylation of wild-type NP was set to 100%. C, Quantification of phosphorylation analyses. D, In vitro kinase assay for CKII. HeLa cells were infected with MVA-T7 and transfected with plasmids encoding NP or NP^regII as described under Figure 1. One-half of cell cultures was metabolically labeled with [³⁵S]methionine and NP was immunoprecipitated (lanes 1, 4, 7, and 10). NP expressed in the nonlabeled cell cultures was immunoprecipitated, dephosphorylated with alkaline phosphatase, and then incubated with [³²P]ATP without (lanes 2, 5, 8, and 11) or in presence of recombinant protein kinase CKII (lanes 3, 6, 9, and 12). Samples separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and analyzed by bioimage analyzer BAS-1000. [³⁵S]methionine-labeled NP served as expression control (lanes 1, 4, 7, and 10). MVA-T7-infected cells and mock control showed no specific signals (lanes 7–12).

The amino acid analysis showed that mainly serine residues were phosphorylated in region II. We replaced all serine residues in the region and analyzed whether the phosphorylation state of the respective mutants was changed. We started with serine 446, which is located close to the serine cluster, and replaced it by alanine (NP^regII446A) (Figures 3A and 3B). We found phosphorylation of NP^regII446A to be reduced by 40%, compared with NP^regII (Figures 3B and 3C). Then, the first 3 serine residues of the cluster were also substituted by alanine (NP^regIIAS), which did not reduce phosphorylation significantly in comparison to NP^regII446A suggesting that the first 3 serines of the cluster were not phosphorylated (Figure 3). Next, all serines in the cluster except serine 454 were substituted by alanine. Serine 454 was substituted by aspartate (NP^regIIAA) to avoid a putative instability of the mutant when all of the anticipated negative charges in the cluster were removed. NP^regIIAA showed a phosphorylation signal, which was reduced, compared with NP^regII, by >90% (Figure 3C). These data indicated that serines at position 453–455 together with the adjacent serine at position 446 represent the major phosphorylation acceptor sites of NP region II.

Figure 3.

Localization of phosphorylation acceptor sites within region II. A, Schematic presentation of NP^regII substitution mutants NP^regII 446A, NP^regII AS, or NP^regIIAA. B and C, Metabolic labeling of NP. HeLa cells were infected with MVA-T7, transfected, and radio-labelled as described under Figure 1. NP and mutants of NP were immunoprecipitated and radioactive signals quantified (n = 4). Phosphosignals were normalized by [³⁵S] methionine signals. Phosphorylation of NP^regII was set to 100%.

Phosphorylation of NP Region II Does Not Influence Inclusion Body Formation and Interaction With VP30 and VP35

NP is the central nucleocapsid protein of MARV. It interacts with itself and the other nucleocapsid proteins, VP35 and VP30, in the process of RNA synthesis and nucleocapsid morphogenesis. To analyze whether phosphorylation of NP region II influenced interaction between nucleocapsid proteins, we constructed full-length NP mutants containing substitutions in region II (Figure 4A). All NP constructs contained a serine-to-alanine substitution at position 446. In addition, either the first 3 serine residues of the cluster were substituted by aspartate and the last 3 serine residues by alanine or vice versa to mimic phosphorylated and nonphosphorylated serine residues in the cluster (NP_AD, NP_DA). In addition, we substituted all serines in the cluster with either alanine or aspartate (NP_AA, NP_DD). The resultant mutants were coexpressed together with VP35 or VP30, and the intracellular localization of the proteins was analyzed. As expected from previous publications, NP formed intracellular inclusions into which VP35 and VP30 were recruited [2]. All tested mutants of NP behaved like wild-type NP, indicating that phosphorylation of region II is neither required in the formation of NP inclusions nor essential for the recruitment of VP35 and VP30 into inclusions, which is considered a key step in the morphogenesis of nucleocapsids (data not shown). To confirm these results, we performed coimmunoprecipitation analyses, which also showed that NP self-interaction and interaction of NP with VP35 was not influenced by phosphorylation of region II (authors' unpublished data).

Figure 4.

Function of region II phosphorylation for viral RNA synthesis and recruitment of nucleocapsids. A, Schematic presentation of full-length NP mutants NP_AA, NP_DD, NP_AD, and NP_DA. Full-length NP carrying alanine and/or aspartate substitutions at the phosphorylation site. B, Scheme of Marburg virus (MARV)–specific iVLP assay using mutants of NP. Plasmids encoding for all viral proteins together with a MARV-specific minigenome (MARV-MG) were transfected into producer cells. NP was replaced by the different phosphorylation mutants, as indicated. Infectious virus like particles (iVLPs) containing either wild-type NP or the respective NP mutant were used for infection of indicator cells that had been previously transfected with plasmids encoding the viral nucleocapsid proteins and the NP mutant, as indicated. C, Analysis of MARV-specific transcription in producer cells. A MARV-specific iVLP assay was performed. When indicated, wild-type NP was replaced by mutants of NP. At 72 p.t., cells were lysed, and Renilla reporter activity reflecting MARV-specific transcription was measured using Dual Luciferase Assay (Promega). Renilla luciferase activity was normalized against firefly luciferase activity reflecting cellular transcription. Activity of wild-type NP was set to 100 % (n ≥ 13). D, Incorporation of NP mutants in infectious virus-like particles. Generated MARV-specfic iVLPs were purified from supernatant of producer cells and submitted to a protease protection assay. NP signals were detected and quantified by Odyssey InfraRed Imager (LiCor). Cell lysate was analyzed as expression control. E, Analysis of MARV-specific transcription in indicator cells. A MARV-specific iVLP assay was performed replacing wild-type NP with mutants of NP. Purified iVLPs were used for infection of indicator cells that had been previously transfected with plasmids encoding the nucleocapsid proteins VP35, VP30, and L. NP was again replaced by NP mutants, as indicated in producer cells. At 60 p.i., cells were lysed, and Renilla reporter activity was measured reflecting MARV-specific transcription. Activity of wild-type NP was set to 100 % (n ≥ 3).

Phosphorylation of NP Region II Modulates Viral Transcription of Replication

NP and the mutants of NP were then used in a MARV-specific infectious virus like particle assay to test their function in MARV-specific transcription and replication (Figure 4) [12]. When the mutants of NP were used in this assay, we found that all of them were able to replace NP in viral transcription/replication. Moreover, mutant NP_AD significantly increased viral RNA synthesis in comparison with wild-type NP (Figure 4). Because of the expression of all viral proteins in the infectious virus like particle assay, mininucleocapsids are incorporated into virus like particles that are released into the supernatant of the producer cells [12]. We therefore analyzed whether mutations in region II of NP had impact on the ability of nucleocapsids to be recruited into virus like particles. We found that all mutants were incorporated into virus like particles as efficiently as NP wild-type (Figure 4). The situation in the producer cells reflects late stages of MARV infection, because the intracellular amount of viral proteins and template is high and allows transcription and replication of viral RNA, as well as morphogenesis and budding of virus like particles [12]. Our data indicated that, at these stages of MARV infection, phosphorylation of the serine cluster in the phosphorylated region II is not critical for interaction of NP with itself, the other nucleocapsid proteins, transcription, and replication. Moreover, phosphorylation of region II was not involved in the phosphorylation-dependent recruitment of NP into viral particles that had been described elsewhere [3].

We were then interested in the role of phosphorylation of NP region II during early stages of infection which are characterized by limited amounts of template. To this end, we produced infectious virus like particles that had NP replaced by the phosphorylation mutants of NP. Infectious virus like particles were purified from the supernatant of producer cells and aliquots were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot to confirm that equivalent amounts of particles were produced (data not shown). The infectious virus like particles were then used to infect HUH7 cells (indicator cells) that had been pretransfected with plasmids encoding VP35, L, and NP or the same mutant of NP that was used to produce the respective infectious virus like particles. At 60 hours after infection, cells were monitored for reporter gene activity. In contrast to the results gained with the producer cells, in the indicator cells, all NP mutants (except NP_AD) were less effective in supporting minigenome activity (ie, viral RNA synthesis than wild-type NP) (Figure 4). Neither mutants that mimic complete dephosphorylation (NP_AA) nor mutants mimicking complete phosphorylation of the serine cluster (NP_DD) were able to fully support RNA synthesis like wild–type NP. Infectious virus like particles containing NP_AD, however, produced ∼1.9 times more reporter gene activity than did infectious virus like particles containing wild–type NP. The effect of NP_AD seems to be more pronounced in the indicator cells than in the producer cells, suggesting that the positive effect of NP_AD on viral RNA synthesis is additive. This could happen if viral transcription and replication are both influenced by NP_AD. Increased replication led to higher amounts of templates in the producer cells and can be recruited by NP into the infectious virus like particles. These particles provide initially more templates for transcription and replication in the indicator cells and produce a higher reporter gene signal. These results are similar to those published for the rabies virus nucleoprotein (N), for which it was shown that changes in phosphorylation had a similar influence on both viral transcription and replication [19].

Reporter gene activity was generally decreased when mutants of NP were used carrying negative charges at the positions of the first 3 serine residues of the serine cluster (NP_DA and NP_DD). This finding is consistent with our observation that these residues are unlikely be phosphorylated in wild-type NP. When all serine residues in region II were replaced by alanine (NP_AA), mimicking a permanently nonphosphorylated region II, the mutant was also impaired in supporting transcription/replication at earlier stages of infection indicating that negative charges in the second part of the serine cluster are important for proper function of NP.

Phosphorylation of rabies virus N has been shown to influence the encapsidation of viral leader RNA [20]. The authors showed that non-phosphorylated rabies nucleoprotein binds strongly to viral RNA and simultaneously the polymerase function seems to be inhibited. It is suggested that the strong binding of NP to RNA inhibited access of the polymerase complex to the template. Upon phosphorylation, the binding of NP to the RNA is weakened, probably as a result of repulsion of the negatively charged RNA and the negative phosphate groups; thus, polymerase might access the RNA template to transcribe and replicate the RNA [20]. This hypothesis is strengthened by the fact that the phosphorylated serine residue of rabies virus N (serine 389) is located close to the RNA binding site (aa 289–352). A similar mechanism can be envisaged to explain the role of phosphorylation of region II of MARV NP.

Taken together, we localized the major phosphorylation acceptor sites in region II of MARV NP and showed that phosphorylation of a characteristic serine cluster in the region II modulates efficiency of viral RNA synthesis, especially during early stages of viral life cycle, when the availability of templates is limited.

Funding

The study was funded by the Deutsche Forschungsgemeinschaft (SFB 593 and SFB 535) and the Schering Foundation to N. B.

The authors wish to thank Angelika Lander and Ulla Thiesen for technical assistance and Winfried Weissenhorn for helpful discussion.

Author notes

Potential conflicts of interest: none reported.

a

These authors contributed equally to this work.

b

Present affiliation: Baxter Bioscience, Vienna, Austria (B. H.); and Charité Berlin, Department of Hepatology and Gastroenterology, Berlin, Germany (H. K.).