Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure
[['Chronic infection with hepatitis B virus ( HBV ) puts more than 250 million people at a greatly increased risk to develop terminal liver disease [1] .', 'HBV , the prototypic hepadnavirus , is a small enveloped virus that replicates its 3 kb DNA genome through capsid-internal reverse transcription of a pregenomic ( pg ) RNA ( reviewed in [2 , 3] ) .', 'The virion envelope consists of a lipid bilayer into which the small ( S ) , middle ( M; PreS2/S ) and large ( L; PreS1/PreS2/S ) surface proteins are embedded [4–6] .', 'Binding of L to the HBV receptor sodium taurocholate cotransporting polypeptide ( NTCP ) is essential for infection ( reviewed in [7] ) ; in addition , L contributes a "matrix domain" that interacts with the capsid for virion morphogenesis ( reviewed in [8] ) .', 'The icosahedral HBV capsid ( core particle ) is composed of 120 dimers ( triangulation number T = 4 ) of a single core protein ( HBc ) species of 183–185 amino acids ( aa ) in length; a minor capsid class ( T = 3 ) comprises 90 HBc dimers .', 'The HBc monomer encompasses an N terminal assembly domain [9] , linked through residues 141–149 [10] to an arginine-rich C terminal domain ( CTD; Fig 1A ) .', 'The CTD is crucial for specific co-encapsidation of a complex of pgRNA and viral polymerase ( P protein ) during replication but it can also mediate non-sequence-specific packaging of RNA ( Fig 1B ) , e . g . when HBc is expressed in E . coli [9 , 11–13] .', 'Structural analyses of such capsid-like particles ( CLPs ) , mostly from CTD-less variants like HBc149 [9] , revealed five α-helices [14–16] .', 'Helices α3 and α4 form an antiparallel hairpin; for dimerization , two such hairpins associate into four-helix bundles which protrude as spikes from the capsid surface ( Fig 1B ) .', 'Helix α5 plus the downstream sequence to position 140 harbor the major inter-dimer contacts .', 'Little is known about the structure of the CTD; in current CLP structures the visible sequence commonly ends within the linker [10 , 14–19] .', 'Regarding CTD disposition most data support a luminal localization [20–23] .', 'However , a permanent internal disposition as well as a static nucleic binding capacity are incompatible with the full set of HBc functions in the viral life-cycle ( Fig 1C ) .', 'Beyond pgRNA/P protein encapsidation these include CTD-mediated reverse transcription [24] into single-stranded ( ss ) minus-DNA and then partly double-stranded ( ds ) relaxed circular ( RC ) DNA [24–26]; capsid envelopment for secretion of virions [8]; and , upon infection of a new cell , transport of the viral genome to the nuclear pore so as to release the RC-DNA into the nucleoplasm [27 , 28] for conversion into covalently closed circular ( ccc ) DNA [29 , 30] .', 'Nuclear transport requires binding of CTD-encoded nuclear localization signals ( NLSs; [31] ) to cytosolic transport receptors [32–35] such that at least one CTD per capsid must become exposed [18 , 27 , 32 , 36 , 37] .', 'Hence the capsid must safely stow ss and also ds nucleic acid with twice as many negative charges and then orderly let go of it .', 'A likely mechanism underlying these CTD dynamics is transient phosphorylation , early-on hinted at by a capsid-associated protein kinase activity [38 , 39] .', 'However , neither this kinase nor other potentially HBc-relevant kinases have unambiguously been identified .', 'Proposed candidates include Ca2+ and/or lipid-activated protein kinase C ( PKC; [36 , 40] ) ; cyclic AMP dependent protein kinase A ( PKA; [41 , 42] ) ; serine/arginine-rich protein kinase 1 and 2 ( SRPK1 , SRPK2; [43] ) ; cyclin-dependent kinase 2 ( CDK2; [44] ) ; and polo-like kinase 1 ( PLK1; [45] ) .', 'Major analytical challenges are the presence in human cells of >500 protein kinases plus >200 phosphatases [46]; the repetitve nature of the HBc CTD sequence ( Fig 1A ) ; the limited specificity of pharmacological kinase inhibitors; and especially the lack of assays that feasibly distinguish non-phosphorylated and differently phosphorylated HBc species .', 'As a surrogate , various studies assessed the impact of serine and/or threonine ( S/T ) replacements in HBc by alanine ( A ) to prevent phosphorylation , and by aspartic acid ( D ) or glutamic acid ( E ) to mimic phosphorylation [47–50] .', 'Collectively these data suggest that phosphorylation is necessary early and some dephosphorylation late during replication .', 'However , genetic mimics cannot model the dynamics of phosphorylation .', 'Phospho-HBc-specific antibodies [51 , 52] are valuable but they interrogate only few of the many options for phosphorylation site occupancy .', 'Correlation with specific replication states is further convoluted by the variety of capsid forms , including nonenveloped capsids [53] and the recently found enveloped genome-less capsids ( "empty virions"; [52] ) which seem to by far outnumber infectious virions ( reviewed in [54] ) .', 'The most direct evidence for the dynamics in core protein phosphorylation comes from duck HBV ( DHBV ) .', 'Intracellular DHBV core protein ( DHBc ) showed up to four distinct phosphorylation-dependent bands in normal SDS-PAGE [55] whereas virion-derived DHBc displayed a single , non-phosphorylated band , as confirmed by mass spectrometry ( MS ) [56] .', 'The presence in virions of mostly mature viral ds DNA [57] , similarly observed for HBV [58] , supported the "maturation signal" hypothesis [57] whereby capsid-internal genome maturation , perhaps sensed by the CTDs and/or their phosphorylation status , exposes interaction sites for L protein on the capsid surface ( Fig 1D ) .', 'The finding that also empty though not ss nucleic acid containing capsids can be enveloped prompted a revised model whereby RNA or ssDNA actively inhibit envelopment [52 , 59] .', 'A cryoEM comparison between recombinant RNA containing capsids and serum virus-derived , supposedly dsDNA bearing capsids had indeed revealed subtle differences [17] .', 'However , at the time empty virions in serum [52 , 60] were not known; hence potential envelopment-relevant structural differences and their correlation with HBc phosphorylation remain open ( Fig 1C , i-iii ) .', 'Some natural HBc mutations such as F97L [61–63] promote secretion of immature , ssDNA containing virions .', 'Such capsid proteins might by default adopt an envelopment-proficient conformation ( Fig 1D ) .', 'Yet , while affecting in vitro assembly [64] no differences in stability or morphology between wild-type and F97L HBc particles were detectable [63 , 65] .', 'However , a potential role of phosphorylation could not be assessed at the time .', 'In sum a large body of data supports correlations between HBc phosphorylation , genome maturation and capsid structure , including CTD disposition and envelopment-competence; however , fundamental information for disentangling these interdependencies is lacking .', 'In order to generate such information we here established the efficient recombinant production of distinct phospho-HBc species and showed the feasibility of Phos-tag SDS-PAGE [66] for their differentiation .', 'Using MS and mutagenesis we identified seven hydroxy amino acid residues in the CTD as target sites for SRPK1 [34 , 43] while PKA and PKC modified fewer , yet to be mapped sites .', 'Full occupancy of the SRPK1 sites massively reduced HBc183 CLP RNA content while abrogation of one phospho-site restored substantial RNA binding .', 'Full phosphorylation also caused differences in CTD accessibility to trypsin , regardless of the F97L mutation .', 'Importantly , in human hepatoma cells the bulk of HBc appeared as highly phosphorylated as SRPK1-coexpressed HBc .', 'These data pave the way for a comprehensive characterization of the substrate properties of hepadnaviral core proteins for individual kinases and phosphatases .', 'Virologically they suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few dephosphorylation events could enable specific packaging of the pgRNA/P protein complex .'], ['Eukaryote-like S/T protein kinases are rare in eubacteria [67] and apparently absent from E . coli laboratory strains .', 'Hence co-expression of HBc with a mammalian kinase should allow the background-free evaluation of its action on HBc .', 'In a first step we created T7 promoter-based vectors carrying an E . coli codon usage adapted HBc gene ( HBc183opt ) to boost expression of full-length HBc183 ( Fig 1A ) .', 'CLP yields were indeed around three-fold higher ( S1 Fig ) compared to the older non-optimized HBc gene [68] in the same pET28a2 vector [69 , 70] , and they increased further using pRSF-Duet derived vectors ( see below ) .', 'For the coexpression approach the recombinant kinase must be enzymatically active and target sites in HBc must not be sequestered by CLP formation .', 'We thus chose human SRPK1 as the first candidate HBc kinase .', 'Beyond its very high affinity for the HBc CTD [34 , 43 , 71] we had already shown HBc phosphorylating activity of the full-length enzyme in bacteria [11]; here we used an N terminally His6-tagged variant , NHisSRPK1ΔNS1 , in which an internal deletion boosts soluble expression in bacteria [72] .', 'To minimize experimental variations seen when HBc and kinase were expressed from two separate plasmids [11] we devised a single-vector dual expression system; it features a Tet repressor / anhydrotetracycline ( AHT ) inducible Tet promoter cassette plus an isopropyl β-D-1-thiogalactopyranoside ( IPTG ) regulatable T7 promoter cassette ( S2 Fig ) .', 'Functionality was shown by inducer-specific expression of eGFP from one and mCherry from the other cassette ( S2 Fig ) .', 'HBc183opt in the T7 cassette was expressed at up to five-fold higher levels than from the pET28a2 vector ( S3 Fig ) .', 'Of note , expression from the Tet cassette was affected by the specific ORF sequence .', 'For instance , the basal level of NHisSRPK1ΔNS1 expression without AHT was still about one third as high as with inducer ( S3A Fig ) ; hence in subsequent SRPK1 coexpression experiments AHT was omitted .', 'Either way , in line with its high affinity for HBc [34 , 43] most of the SRPK1 cosedimented with the CLPs , both from wild-type and F97L HBc ( S3 Fig ) ; separation was achieved by immobilized metal ion chromatography ( IMAC ) under semi-denaturing conditions ( S4 Fig ) .', 'Altogether , the system provided easy access to milligram amounts of CLPs per 100 ml of induced bacterial culture .', 'Low level co-expression in E . coli of full-length SRPK1 with HBc183 had generated a mixture of HBc species containing from one to several phosphoryl groups [11]; this prompted the follow-up use of a triple S>E variant , HBc183-EEE ( S155E_S162E_S170E ) as a homogeneous genetic mimic of such partially phosphorylated HBc183 [34 , 35] .', 'HBc183-EEE CLPs contained ~25% less RNA than wild-type HBc183 CLPs [73] .', 'To evaluate the impact on CLP RNA content of the much higher NHisSRPK1ΔNS1 expression in our new system we separated the CLPs by NAGE , then stained packaged RNA by ethidium bromide ( EB ) and the protein shell by Coomassie Brillant Blue ( CB ) [9 , 74]; other stains , e . g . Sybr Green 2 ( SG2 ) for RNA and Sypro Ruby ( SR ) for protein [75] , could be used instead ( see below ) .', 'The intensity of RNA vs . protein stain per band is proportional to the CLP´s RNA content .', 'Fig 2A shows such an analysis for HBc183_F97L expressed in the absence ( -SRPK ) vs . presence of SRPK1 .', 'Despite much weaker EB staining the +SRPK1 samples showed even stronger CB staining; in either case abundant CLPs were visible in negative staining EM; wild-type HBc183 CLPs gave comparable results .', 'To appreciate the extent of reduction in RNA content caused by NHisSRPK1ΔNS1 coexpression we used a reference set of CLPs from C terminally truncated HBc proteins , starting with the CTD-less variant HBc140 .', 'SG2 and SR allowed semiquantitative assessment of the relative fluorescence intensities of each band ( Fig 2B ) using a laser scanner ( Typhoon FL7000 , GE Healthcare ) .', 'The ratio of RNA: protein fluorescence for non-phosphorylated HBc183 CLPs was set to 100% , to which the ratios for the other CLPs were normalized .', 'Accordingly , SRPK1-coexpressed HBc183 CLPs contained nominally 27% as much RNA as the HBc183 reference CLPs , very similar to CTD-less HBc140 CLPs .', 'For the other variants , the ratios increased with increasing CTD length , in accord with the electrostatic "charge balance" hypothesis [13 , 26 , 76] .', 'An estimate of absolute CLP RNA content as molar ratio of nucleotides ( N ) per core protein ( P ) subunit ( N/P ) was obtained from UV-VIS spectra by deconvoluting the superimposed RNA and protein absorbances [73]; spectra were recorded using gradient-enriched CLPs incubated with 1/100 the amount of ( w/w ) of RNase A and subsequently dialyzed to remove non-packaged RNA .', 'For HBc183 CLPs we obtained ( N/P ) values ± standard deviation ( SD ) of 16 . 4±2 . 2 ( n = 8 ) ; for a T = 4 CLP this corresponds to an RNA content equivalent to ~4 , 000 nt , similar to other reports [12 , 73] .', 'Much in contrast , SRPK1-coexpressed HBc183 CLPs gave a ratio of only 2 . 3±0 . 5 ( n = 8 ) , not significantly different from CTD-less HBc140 ( 2 . 9±1 . 3; n = 4 ) and HBc149 CLPs ( 1 . 8±1 . 5; n = 4 ) .', 'Intermediate RNA contents were found for HBc161 , 163 , 166 and 173 CLPs , as shown in Fig 2C in comparison with the NAGE/SG2:SR procedure .', 'While the apparent overestimation of low RNA contents by the latter method could later be reduced by several improvements ( Materials and Methods ) , both data sets corroborated that SRPK1 coexpression reduced RNA content as strongly as deletion of the entire CTD .', 'This implied a phosphorylation extent sufficient to shift assembly to an RNA-independent , protein-protein interaction driven mechanism [37 , 77] , perhaps by neutralizing most of the positive CTD charges [13] .', 'The Phos-tag chelator coordinated with two Me2+ ions binds to phosphoryl groups , including in proteins [78] .', 'In SDS-PAGE gels containing copolymerized Phos-tag acrylamide and loaded with Mn2+ or Zn2+ phospho-proteins migrate more slowly than their non-phosphorylated forms [66] .', 'Hence this technique appeared attractive to detect HBc phosphorylation .', 'After optimization we routinely used home-made gels containing 75 μM Phos-tag acrylamide loaded with 150 μM Mn2+ .', 'As shown in Fig 3 , in normal SDS-PAGE both HBc183 and HBc183_F97L migrated to the same ~21 kDa position , regardless of SRPK1 coexpression .', 'In Phos-tag SDS-PAGE , however , exclusively the kinase-coexpressed proteins exerted a drastically reduced mobility .', 'Band ladders produced in in vitro SRPK1 phosphorylation and lambda phosphatase dephosphorylation reactions with a non-assembling GFP-CTD fusion protein suggested phosphorylation at five or more positions .', 'Analogous coexpression of HBc183 with the catalytic domains of PKA ( Genbank NP_032880 . 1 ) and PKC theta ( Genbank NP_001269573 . 1 ) , chosen owing to their reportedly functional expression in E . coli [79 , 80] , caused a less pronounced retardation ( see below ) .', 'The number of phosphoryl groups introduced into HBc183 by SRPK1 was analyzed by matrix-assisted laser desorption/ionization ( MALDI ) time-of-flight ( TOF ) MS . The major MH+ peaks in the relevant 21 kDa region of the spectra had mass/charge ( m/z ) values of 21 , 103 for mono-expressed HBc183 and 21 , 664 for SRPK1-coexpressed HBc183 .', 'These values match most closely to the calculated masses of 21 , 116 Da for completely unmodified HBc183 , and to 21 , 676 Da for a seven-fold phosphorylated HBc183 ( S5 Fig; each phosphoryl group contributes ~80 Da ) .', 'Notably , for SRPK1-coexpressed HBc140 and HBc149 the main peaks still corresponded to the non-phosphorylated proteins; hence the assembly domain and the linker sequence lack SRPK1 target sites .', 'For more accurate mass determination we devised a method ( S1 protocol ) to isolate just the CTD peptide from a tobacco etch virus ( TEV ) protease cleavable NHisGFP-TEV-CTD fusion protein ( Fig 4A ) , expressed with or without kinase .', 'Cleavage reduces the relevant masses to ~5 , 000 Da , with a synthetic N-acetylated CTD peptide serving as reference ( Fig 4B ) .', 'Methods development included replacement of the C terminal Cys residue by Ala ( C183A in full-length HBc numbering ) .', 'In brief , the NHisGFP-TEV-CTD protein was enriched by Ni2+ IMAC under semi-denaturing conditions , then incubated with His-tagged TEV protease [81] to release the CTD peptide; all His-tagged components ( uncleaved GFP fusion protein , the GFP-containing cleavage product and TEV protease ) were removed by another round of IMAC .', 'The clipped-off CTD peptide in the flow-through was concentrated and further enriched by ultrafiltration .', 'An SDS-PAGE analysis of the final products obtained upon coexpression with SRPK1ΔNS1 or the PKA catalytic domain ( PKAcd ) or without kinase , alongside the synthetic CTD peptide , is shown in Fig 4C .', 'Importantly , MS of the SRPK1 sample ( Fig 4D ) showed one major peak with excellent agreement to a seven-fold but not six- or eight-fold phosphorylated CTD peptide .', 'Hence there are seven SRPK1 target sites in the HBc sequence 146–183 .', 'The m/z values for the synthetic and the non-phosphorylated fusion protein-derived CTD peptide were <1 Da off the calculated masses ( S6 Fig ) .', 'The PKA-coexpressed sample showed two main peaks , matching four and five phosphoryl groups , respectively; for full-length HBc183 as substrate the products from PKA and PKC coexpression carried only three phosphoryl groups ( S7 Fig ) , possibly due to assembly-mediated target site sequestration .', 'To identify the seven SRPK1 sites in HBc we individually mutated the seven S residues plus one T residue in the CTD ( S155 , T160 , S162 , S168 , S170 , S172 , S178 and S181 ) to A , coexpressed the mutant proteins with SRPK1 and monitored phosphorylation by MS ( Fig 5A ) and by immunoblotting after Phos-tag SDS-PAGE .', 'For all mutants but one MS revealed a best match to the six-fold phosphorylated forms , i . e . loss of one phosphorylation site .', 'The exception was mutant S181A which still matched best to a seven-fold phosphorylated species .', 'Hence all hydroxy amino acids in the HBc CTD including T160 yet except S181 are substrates for SRPK1 .', 'Double and triple S/T>A mutants confirmed the data as the number of lost phosphorylation sites always corresponded to the number of S/T replacements by A ( or by G or R ) , except when S181 was mutated ( S7 Fig ) .', 'In line with their at least six-fold phosphorylation all single S/T variants were similarly strongly retarded in Phos-tag SDS-PAGE as SRPK1-coexpressed wild-type HBc183 , as shown by immunoblotting ( Fig 5B ) with the assembly domain-directed anti-HBc mAb 1D8 [82] .', 'Notably , though , the correlation between the number of phosphorylation sites and mobility was not strictly linear .', 'The six-fold phosphorylated variants S155A , S176A , and S178A migrated about as slowly as the seven-fold phosphorylated wild-type protein and variant S181A .', 'In contrast , the likewise six-fold phosphorylated variants T160A , S162A and S170 ( marked with asterisks in Fig 5B ) had distinctly higher mobilities .', 'Hence beyond the number of phosphoryl groups also their sequence context contributes to the interaction with the Phos-tag .', 'Knowledge of the S/T>A variants´ phosphorylation status also enabled a more detailed characterization of the epitope requirements of mAb T2212 ( see below ) .', 'We then evaluated the impact of the S/T>A mutations on CLP RNA content by the NAGE based SG2/SR staining assay ( Fig 5C , S8 Fig ) but using a more elaborate protocol ( Material and Methods ) .', 'Owing to the lower background signals the relative RNA contents of HBc140 , HBc149 and SRPK1-coexpressed HBc183 as well as HBc183_S181A CLPs were now estimated to ≤10% of that in unmodified HBc183 CLPs ( Fig 5C ) .', 'The mostly three-fold phosphorylation by PKA and PKC caused only minor reductions ( significant only for PKC ) .', 'Remarkably , preventing phosphorylation at one of the seven SRPK1 sites was sufficient to increase RNA contents to generally ~20–30% of non-phosphorylated HBc183 CLPs; additional phosphorylation site mutations further increased RNA content ( S8 Fig ) .', 'Intriguingly , the single S170A mutation restored RNA content to ~50% of unmodified wild-type HBc183 CLPs , and this difference to the other single variants was significant ( p<0 . 001; n = 6 ) .', 'Hence phosphorylation of S170 may be particularly effective in suppressing RNA binding by the HBc CTD .', 'HBc183 CLPs are sensitive against SDS [9 , 65]; hence titration with SDS might reveal whether high-level phosphorylation and/or the concomitant low RNA content affect CLP stability .', 'To this end we incubated HBc183 CLPs and HBc183_F97L CLPs expressed in the absence vs . presence of SRPK1 with either 1x TAE electrophoresis buffer ( Ø ) , or a native glycerol-based DNA loading buffer ( Ø* ) , or increasing amounts of a 6x DNA loading buffer ( NEB Purple ) containing 0 . 48% SDS ( 0 . 08% at 1x concentration ) .', 'RNA and protein were monitored by EB and CB staining; expectedly , SRPK1-coexpressed CLPs showed only faint EB versus CB signals ( Fig 6 , right panels ) .', 'At 0 . 024% SDS the nonphosphorylated CLPs released some of the RNA ( faster migrating EB smear ) although the major band remained at the original position .', 'At 0 . 048% SDS , the fast RNA smear increased , while the original CLP band disappeared in favor of a slower , less intense band from which EB signals emanated upwards to the cathode; still higher SDS concentrations enhanced these effects .', 'CB staining of the latter bands suggests they may represent complexes of HBc183 proteins with exposed CTDs ( forced to move towards the cathode ) to which some of the initially encapsidated RNA is bound .', 'In accord with previous data [65] no clear difference was detectable for wild-type vs . F97L CLPs .', 'Interestingly the SRPK1-phosphorylated low RNA CLPs also showed a sharp mobility transition between 0 . 024% and 0 . 048% SDS , although the new products migrated a bit slower than those from the nonphosphorylated CLPs .', 'In sum , SDS sensitivity of the HBc CLPs was neither affected by seven-fold phosphorylation of the CTD , nor by RNA content or the F97L mutation .', 'The high arginine content makes the entire HBc CTD an excellent substrate for trypsin; however , protease accessibility is affected by the CLP structure [37 , 65 , 83] .', 'In our hands , trypsin had converted part of the unmodified wild-type HBc183 into a major product with HBc149-like mobility [83] .', 'To reveal potential alterations of this pattern by phosphorylation , RNA content and/or the F97L mutation , we monitored the kinetics of trypsin action on non-phosphorylated vs . phosphorylated CLPs from wild-type and F97L HBc183 protein .', 'First we addressed a potential inhibition of trypsin cleavage by CTD phosphorylation by comparing the trypsin sensitivity of the non-assembling NHis-GFP-TEV-CTD fusion protein ( see Fig 4A ) expressed in the absence vs . presence of SRPK1 .', 'Both proteins were completely cleaved with similar kinetics ( Fig 7A , left panel ) .', 'Under the same conditions , HBc183 in non-phosphorylated CLPs was only partially cleaved within 30 min ( Fig 7A , right panel ) , with about half the molecules remaining intact and the other half displaying HBc149-like mobility; this pattern remained stable over 2', 'h . Higher temporal resolution ( Fig 7B ) revealed the transitory formation of intermediate mobility bands which had mostly disappeared at 30 min in favor of the stable HBc149-like product ( labeled "4" ) ; thereafter the ratio between presumably intact HBc183 ( labeled "1" ) and product "4" remained constant for at least 4', 'h . NAGE did not reveal significant alterations in EB vs . CB staining over time ( Fig 7B , lower panels ) .', 'Hence loss of half the CTDs affected neither RNA content nor surface charge , indicating the overall CLP structure remained intact .', 'Comparable results with HBc183_F97L CLPs ( Fig 7C ) ruled out major effects of the F97L mutation .', 'In contrast , SRPK1-coexpressed HBc183 CLPs ( Fig 7D , top panel ) presented two additional discrete , stable products ( "2" and "3" ) of which product 3 eventually reached similar levels as the remaining full-length protein .', 'By comparison with HBc marker proteins ( Fig 7D , lower panel ) products 3 and 2 resulted from cleavage between positions 157–159 and 166–172 , respectively .', 'HBc_F97L CLPs produced a very similar four-band pattern in SDS-PAGE ( Fig 7E , top panel ) ; in NAGE CLP mobility and RNA content again remained unaltered ( Fig 7E , bottom panels ) .', 'While quantitation of the band 1 to 4 proportions could be disturbed by the presence of not fully intact CLPs , formation of bands 2 and 3 exclusively from the phospho-CLPs strongly implies distinct steric constraints for the band 2 and 3 processing sites compared to unmodified CLPs .', 'Scenarios compatible with the data ( complete CTD extrusion vs . looping out of CTD parts ) are shown in S9 Fig . To directly assess the impact of seven-fold CTD phosphorylation and/or low RNA content on capsid structure we analyzed nonphosphorylated vs . SRPK1-phosphorylated HBc183 CLPs by cryoEM ( S10A and S10 Fig ) and calculated image reconstructions ( Fig 8 ) at 7 . 8 Å ( B = 707 Å2 ) and 7 . 9 Å ( B = 979 Å2 ) resolution ( S10C Fig ) .', 'Most striking was a distinct shell of internal density exclusively in the non-phosphorylated particles ( Fig 8B ) which must represent CTDs plus packaged RNA .', 'The relatively high B-factors suggested some structural heterogeneity within each CLP class , hence the gross features in the shells of unmodified vs . phosphorylated particles appeared very similar .', 'However , in defined areas significantly different grey value distributions occurred ( Fig 8C; see S2 Protocol for significance calculations ) .', 'For the SRPK1 coexpressed CLPs they could be best described as reduced order in the N terminal regions which flank the spikes ( Fig 8C bottom vs . top: less red color around the three-fold axes ) , plus an apparent stretching of the spike helices and the inter-dimer contact regions ( Fig 8C bottom vs . top: more blue color within the dimer contours ) .', 'To improve resolution for fitting these differences into atomic models of HBc we vitrified additional aliquots of the same sample preparations which , fortuitously , had been stored for 1 . 5 years at 4°C .', 'Corroborating the high stability of HBc CLPs , NAGE , SDS-PAGE and Phos-tag SDS-PAGE revealed no signs of degradation or loss of phosphorylation .', 'Surprisingly , however , by 3D-classification the majority of these "aged" CLPs , especially the phosphorylated ones , grouped into a distinct class from the bulk of fresh particles ( vitrified within two weeks post preparation ) used above .', 'The aged particle reconstructions had higher resolution and lower B-factors ( 6 . 3 Å and 6 . 6 Å , and 555 Å2 and 466 Å2 , respectively , for non-phosphorylated vs . SRPK1-phosphorylated CLPs ) , in line with increased ordering with time .', 'For the non-phosphorylated CLPs the differences were only minor but for the phospho-HBc particles they were pronounced .', 'As in the comparison with fresh unmodified CLPs ( Fig 8C ) the freshly vitrified phospho-CLPs showed extended spikes and less ordered N termini ( S11A Fig , top ) whereas after ageing they appeared very similar to the non-phosphorylated ones ( S11A Fig , bottom ) .', 'This suggests a conformational maturation process , although not necessarily in the classic sense of gaining envelopment competence .', 'Fitting the HBc149-derived crystal structure 1QGT [16] into these reconstructions accounted for most density in the assembly domains but left unaccounted internal density close to the inner CLP surface , likely related to CTDs plus RNA in the non-phosphorylated CLPs yet only CTDs in the phospho-CLPs .', 'The undisturbed view onto the inner shell surface of the latter particles revealed extensive tube-like structures around the 5-fold symmetry axes ( Fig 8D , bottom vs . top ) .', 'Connecting to the last visible residues in the crystal structure they probably represent residues from the linker plus the CTD .', 'The respective density extended towards the feet of the spikes , with apparently direct contacts to R112 in both fresh and matured CLPs ( S11B Fig ) .', 'Their visibility implies rather stable interactions , perhaps including electrostatic binding between R112 and phosphorylated CTD residues in the phospho-CLPs ( S9C and S9D Fig ) .', 'Similar though less prominent extra density was also seen in the non-phosphorylated CLPs , although their high RNA content makes a clear distinction between nucleic acid and protein residues difficult ( S11B Fig ) .', 'Recent data suggest that most HBc in mammalian cells from replicating as well as non-replicating vectors is phosphorylated [13 , 52] yet the number of phospho-sites per HBc monomer is unknown .', 'We therefore analyzed HBc in intracellular capsids from the stable HBV producing TetOFF HepG2 . 117 line [84] by Phos-tag SDS-PAGE , using our recombinant phospho-HBc183 proteins as markers .', 'To minimize band distortions seen with crude cytoplasmic lysate we first enriched capsids by sedimentation through 10–60% ( w/v ) Nycodenz gradients ( Fig 9A; S12A Fig ) .', 'The presence of HBV-typical replicative DNA intermediates was corroborated by Southern blotting ( Fig 9B ) ; however , less vs . more mature species were barely separated and the presence of empty particles [54] remains to be addressed .', 'Regardless of this , Phos-tag SDS-PAGE showed a comparably strong retardation of the bulk HBc signals as for SRPK1-phosphorylated recombinant HBc183 ( Fig 9C , short exposure ) .', 'Longer exposure revealed several very weak intermediate mobility bands in the HepG2 . 117 samples , similar to those seen for CLPs coexpressed with PKA and PKC , and for non-phosphorylated CLPs; however , >95% of the band intensity was concentrated in the slowest migrating band .', 'Though this would be in line with an SRPK1-like phosphorylation further investigation using the phospho-CTD specific mAb T2212 [51] indicated a non-identical phosphorylation pattern .', 'As reported mAb T2212 did not recognize unmodified recombinant HBc183 , yet it reacted strongly with the PKA- and some of the PKC-phosphorylated HBc species .', 'Interestingly , fully SRPK1-phosphorylated HBc gave almost no signal whereas single alanine replacements of the phospho acceptor sites S162 , S168 and , weakly , S176 and S178 but not S170 restored reactivity ( S12 Fig ) .', 'Hence the T2212 epitope appears to depend on the presence of phosphoryl groups at some positions , including S170 , yet their absence from nearby sites .', 'To assess whether mAb T2212-reactive phospho-HBc species are present in enveloped particles we separated extracellular particles from HepG2 . 117 cells and from a new HBc183-only expressing Huh7 line by NAGE; recombinant HBc183 CLPs served as control ( Fig 9D ) .', 'Immunoblotting revealed a fast migrating band in the human cell-derived samples which corresponded to naked capsids , as indicated by comigration with bacterial HBc183 CLPs; as before , the latter did not react with mAb T2212 but were detected by the anti-assembly domain mAb 312 ( marked by ** ) .', 'Importantly , mAb T2212 revealed a second , slower migrating band exclusively in the HepG2 . 117 samples which also stained with the anti-HBs mAb 9H9 [85] .', 'Hence capsids containing phosphorylated HBc can be enveloped .', 'More detailed conclusions will require separation from the naked capsids and efficient fractionation of enveloped capsids with differing nucleic acid content .'], ['Our MS data confirmed the absence from E . coli BL21 cells of enzymes capable of phosphorylating any residue in HBc183 ( Figs 3 and 4; S5 and S6 Figs ) .', 'Hence all previously reported properties of E . coli derived HBc CLPs relate to the fully unphosphorylated state .', 'Vice versa , co-expressing a eukaryotic kinase then allows to single out how that specific kinase acts on HBc , as shown here for SRPK1 and per proof-of-principle for PKA and PKC .', 'There are still caveats for interpretation , including the concentration and ratio of kinase to HBc substrate in the bacteria , plus the potential seque
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Hepatitis B virus ( HBV ) replicates its 3 kb DNA genome through capsid-internal reverse transcription , initiated by assembly of 120 core protein ( HBc ) dimers around a complex of viral pregenomic ( pg ) RNA and polymerase .', 'Following synthesis of relaxed circular ( RC ) DNA capsids can be enveloped and secreted as stable virions .', 'Upon infection of a new cell , however , the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular ( ccc ) DNA .', 'HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain ( CTD ) with intrinsically strong non-specific RNA binding activity .', 'Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events .', 'However , neither the relevant enzymes nor their target sites in HBc are firmly established .', 'Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc .', 'Combining Phos-tag gel electrophoresis , mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 ( SRPK1 ) ; fewer sites were phosphorylated by PKA and PKC .', 'Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility , without major structure changes in the capsid shell .', 'The bulk of capsids from human hepatoma cells was similarly highly , yet non-identically , phosphorylated as by SRPK1 .', 'While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex .', 'The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target .']
['The liver-pathogenic hepatitis B virus ( HBV ) is a small enveloped DNA virus that replicates through reverse transcription of a pregenomic ( pg ) RNA .', 'This requires specific encapsidation of pgRNA and viral polymerase into a shell of 240 core protein ( HBc ) subunits .', 'Capsid-internal formation of relaxed circular ( RC ) DNA enables the particle to leave the cell as stable virion; yet , when infecting a new cell it must release the RC-DNA for conversion into another , plasmid-like DNA that templates new viral RNAs .', 'This up and down in nucleic acid interactions is presumably regulated by transient phosphorylation of HBc , mainly in its arginine-rich C terminal domain ( CTD ) which displays strong non-sequence-specific RNA binding .', 'However , neither the phosphorylation sites nor the relevant enzymes are well defined .', 'We developed a recombinant system to produce kinase-specific phospho-HBc species , and adapted a feasible gel assay for their separation .', 'By mutagenesis and mass spectrometry we identified seven target sites for a major candidate kinase , SRPK1 , in the CTD .', 'As full SRPK1 phosphorylation thwarted non-specific RNA binding the comparably high phosphorylation of HBc in human cells suggests how specific pgRNA encapsidation might be achieved .', 'Our new tool set will facilitate disentangling the role of HBc phosphorylation in HBV infection and exploiting it as potential therapeutic target .']
['phosphorylation', 'medicine and health sciences', 'pathology and laboratory medicine', 'enzymes', 'pathogens', 'microbiology', 'enzymology', 'viral structure', 'hepatitis b virus', 'phosphatases', 'serine proteases', 'viruses', 'rna structure', 'proteins', 'medical microbiology', 'microbial pathogens', 'hepatitis viruses', 'viral packaging', 'recombinant proteins', 'viral replication', 'molecular biology', 'virions', 'biochemistry', 'rna', 'post-translational modification', 'nucleic acids', 'virology', 'viral pathogens', 'biology and life sciences', 'proteases', 'organisms', 'macromolecular structure analysis']