Modulation of early host innate immune response by a Fowlpox virus (FWPV) lateral body protein

The avian pathogen, fowlpox virus (FWPV) has been successfully used as vaccine vector in poultry and humans but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication permissive and non-permissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, non-essential FWPV gene knock-out mutants revealed that FPV184 confers immunomodulatory capacity. We report that FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 hours post-infection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies and capable of supressing IFN induction early during the next round of infection.


Introduction 38
Poxviruses are large, enveloped, double-stranded DNA viruses capable of causing 39 disease in mammals, birds and insects. Binding and entry of poxviruses into vertebrate 40 cells is an efficient process for a wide range of cell types, irrespective of the host 41 species, with any host range restriction occurring after viral entry 1 . The complex 42 replication cycles of poxviruses take place exclusively in the cytoplasm although it has 43 long been suggested that poxviruses must interact with host nuclei for productive 44 infection 2-6 . Perhaps the best-studied antiviral host restriction mechanism is interferon 45 (IFN)-mediated, against which almost all viruses have evolved defence 46 mechanisms 7,8 . Some of the first viral anti-IFN defence mechanisms were elucidated 47 using VACV, which expresses multiple inhibitors of IFN induction, JAK/STAT 48 fact that only 110 (42%) of FWPV genes share significant similarity to those in other 74 poxviruses 21 . To identify the innate immunomodulatory factors encoded by FWPV, we 75 previously conducted two broad-scale pan-genome analyses of FP9, a highly 76 attenuated strain used as a vaccine vector in both poultry and mammals 21,22 . In the 77 first study, we identified FPV012 as a modulator of IFN induction by screening a knock-78 out library of 65 non-essential FP9 genes 20,23 . In the second study, using a gain-of-79 function approach in which 4-8 kbp fragments of FP9 were introduced into modified 80 vaccinia Ankara (MVA), we found that FPV014 contributes to increased resistance to 81 exogenous recombinant chicken IFN-α 24 . 82 In this report, we used our existing FP9 knock-out library 20 to screen infected primary 83 chicken embryo fibroblasts (CEFs) for FWPV genes that modulate the induction of 84 interferon-regulated genes (IRG)s. Using this approach, we identified FPV184 as a 85 third FWPV immunomodulatory protein blocking the induction of innate immune 86 responses. Intriguingly, unlike the FWPV immunomodulators FPV012 and FPV014 87 (which are both early viral proteins), FPV184 was found to be a late, structural protein 88 with a functional nuclear localisation signal. Consistent with its ability to modulate ISG 89 responses soon after infection and long before de novo production, we show that 90 FPV184 is packaged into FWPV particles where it resides in the LBs. These results 91 suggest that the packaging of late immunomodulatory proteins, and their subsequent 92 delivery into the nucleus of newly infected cells serve as an immediate early innate 93 immune evasion strategy. 94 95 Results 96

Identification of immunomodulatory signature induced by FWPV∆184 97
We infected CEFs (three independent batches) with 59 individual FWPV mutants, 98 each deficient in one non-essential gene 20,23 . Gene expression was analysed at 16 h 99 This late expression of FPV184 is unusual, if not currently unique, for a non-essential 149 poxviral gene with early immunomodulatory function. The only known example of an 150 immunomodulatory poxviral protein with an early effect is the essential VACV H1 151 phosphatase, which is packaged into poxvirus LBs and delivered into host cells during 152 virus entry to mediate early suppression of STAT1 signalling 17 . Thus, we asked if 153 FPV184 is packaged into FWPV virions and, if so, where in the virions it is located. 154 Purified GFP184 virions (expressing FPV184 with GFP fused to its N-terminus) were 155 left untreated or were subjected to fractionation using NP-40 and/or DTT to separate 156 viral membranes from cores and their associated LBs 26 . Immunoblots directed against 157 FWPV core protein FPV168, and membrane protein FPV140 were used to validate 158 the fractionation 27 . Immunoblots directed against GFP indicated that GFP184 is 159 packaged in virions (Fig. 3d). In untreated and NP-40-treated virions, very little 160 GFP184 was released from virions, suggesting that the protein is not on the virion 161 surface. Treatment with NP-40 and DTT 28 resulted in a 50/50 distribution of GFP184 162 between virion membrane and core/LB fractions, suggesting that GFP184 resides 163 between the viral membrane and the viral core. 164

165
To more accurately define the sub-viral localisation of FPV184 we used correlative 166 super-resolution light and electron microscopy (CSRLEM). Purified GFP184 virions 167 were imaged using stochastic optical reconstruction microscopy (STORM) followed by 168 transmission electron microscopy (TEM). Images were registered to correlate the 169 fluorescence signal with EM structural information ( Fig. 3e; Supplementary Fig. S3 It has been previously reported that VACV A19 and its orthologs, including FPV184, 211 contain 3 highly conserved motifs: Two CxxC motifs in the middle of the protein (amino 212 acids 37-40 and 72-75), whose mutation resulted in a reduction in virus yield, and a 213 basic NLS (FWPV amino acids 9-14: KKRKKR; Fig. 5a and Supplementary Fig. S5) 214 at its N-terminus 30 . While A19 was shown to display nucleo-cytoplasmic localization 215 during infection, mutation of the NLS had no apparent defect on virus growth 30 . 216

217
To study the cellular localisation of FPV184 during infection, a construct expressing 218 GFP/cMyc-tagged FPV184 under the control of an early/late synthetic poxvirus 219 promoter was generated 31 . Consistent with the VACV A19 data, expression of this 220 tagged version of FPV184 in FP9-infected CEFs showed its presence throughout the 221 cytoplasm but concentrated within the nucleus (Fig. 5b). A non-fused EGFP control, 222 was fairly evenly distributed throughout the cytoplasm and nucleus. Expression of V5-223 His-tagged FPV184 from a eukaryotic expression vector, in the absence of infection, 224 showed the same distribution (Fig. 5b). 225

226
To determine whether the identified FPV184 NLS motif was responsible for this 227 nuclear accumulation, we transiently expressed FPV184 lacking its NLS ( Fig. 5c; NLS -228 ). To determine if the nuclear localisation signal found in FPV184 is functionally 229 conserved with VACV A19, we also swapped this region of FPV184 with the 230 corresponding sequence from VACV WR A19 (KSRKKKPKTT) ( Fig. 5c; A19 NLS). its ability to suppress the promoter (Fig. 5d). Consistent with its conserved sequence, 248 VACV A19 NLS was able to rescue suppression by FPV184 A19 NLS to the level 249 observed with FPV184 (Fig. 5d). Since A19 is a late protein and has an essential role in virion morphogenesis, A19 273 knock-out viruses were not constructed and thus the potential role of A19 as an 274 immunomodulator was not evaluated. Whilst conservation of A19 and FPV184 275 suggests an important function for the latter, we were able to generate FWPV lacking 276 FPV184 using two different methodologies, showing that the gene is non-essential for 277 replication in vitro, albeit with a reduced replication rate. For example, FPV138 is a homologue of VACV H1, a protein-tyrosine phosphatase 300 that resides in the LBs, which, upon release into the cytoplasm, dampens type I IFN-301 induced, STAT1-mediated signalling. ISG expression was higher in cells infected with 302 FWPV∆012 than with FPV∆184, in terms of both numbers and expression levels; there 303 was, however, a significant overlap between the ISGs induced by both viruses (Fig.  304 1b). Furthermore, when FPV012 and FPV184 were transiently co-expressed in DF-1 305 cells, they were not synergistic in inhibiting the pI:C-mediated induction of IFN-β 306 The putative role of FPV184 in blocking immediate-early host immune response is 313 also supported by our demonstration, with super resolution microscopy and CSRLEM, 314 that the protein is packaged within the LB and outside the confines of the viral cores 315 where early viral transcription is executed. LB have been described as a poxvirus 316 mechanism for delivery of viral proteins to the cytoplasm of the cell soon after fusion 317 of the MV membrane with cellular plasma or endosomal membranes; their release and 318 disaggregation is believed to depend upon reduction and proteosomal activity 17 . 319 Parallels exist with the herpes virus tegument, which is also located inside the virion, 320 under the envelope but outside of the capsid, and is known to deliver virion host 321 effector proteins into cells 17,37 . Our hypothesis is that FWPV LB contain additional 322 packaged immunomodulatory factors that can act before core activation and early 323 gene expression, to establish a favourable environment in the cytoplasm. Other 324 proteins found in LB in VACV 17 include the phosphoprotein F17 and oxidoreductase 325 G4, both involved in morphogenesis, as well as the dual-specificity phosphatase H1 326 discussed above (FWPV orthologues, FPV103, FPV077 and FPV138, respectively). 327 H1 has immunomodulatory function by virtue of its ability to target STAT-mediated 328 signalling, required for IFN-mediated induction of ISG expression. Our demonstration 329 that FPV184 and VACV A19 are also packaged in LB, with functional evidence that 330 they both have immunomodulatory function, shows that they could complement H1, 331 primarily by blocking induction of IFN but, to a lesser extent, also by blocking IFN-332 mediated induction of ISGs. protein containing an identifiable and functional NLS. Furthermore, the nuclear 343 localisation of FPV184 was found to influence its immunomodulatory ability. Using a 344 luciferase assay, we showed that a construct expressing FPV184 without the NLS only 345 partially abrogated the ability of the protein to inhibit the IFN-mediated induction of 346 Mx1 promoter (Fig. 5d). Whether nuclear localization is partially or fully required for 347 the immunomodulatory ability of FPV184 was unclear as, due to its small size (9.5 348 kDa), low levels of the protein can enter the nucleus without an NLS, via passive 349 diffusion. Myxoma virus encodes a protein termed myxoma nuclear factor (MNF) 42 , an 350 ankyrin repeat containing protein that localises to the nucleus in the absence of an 351 NLS and sequesters NFkB. The cowpox virus protein crmA, even though it is small 352 enough to shuttle between the nucleus and cytoplasm by passive diffusion, requires a 353 leucine-rich nuclear export signal (NES) for its nuclear export 43 . It is possible that, in 354 the presence of the active NLS, the accumulation of FPV184 in the nucleus is 355 dependent on its lack of a NES. 356 Although the conserved block of lysines and arginines found at the N terminus of 357 At different times p.i., the extracellular medium was collected, and the cells were 392 overlaid with 1 ml of fresh medium and stored at -70°C. Intracellular and extracellular 393 viruses were subjected to titer determination by plaque assay 46 . Plaque sizes between 394 wild-type and knock-out viruses were evaluated as before. Briefly, plaques areas were 395 digitally enlarged and calculated in arbitrary units using ImageJ v.32 image analysis 396 software (Fig. 2d). Scatterplots were created with GraphPad prism (Fig. 2e). 397 398

Construction of plasmids and recombinant viruses 399
Construction of FPV184 knock-out virus was done using two different approaches: a 400 PCR mediated knock-out with a guanine-phosphoribosyltransferase (GPT) insertion 401 in the middle of the gene (FWPV∆184, which was used for the microarray study) and

Isolation of FPV184 deletion mutants by PCR-mediated knock-out 414
All primers used in the study are listed in the Supplementary

Isolation of FPV184 deletion mutants by transient dominant selection 428
The FPV184 deletion plasmid was constructed using the previously described vector

Generation of GFP recombinant viruses 447
The FPV184 gene was amplified by PCR with M2952 and M2951. The product was 448 digested with XmaI and SacII (within M2952 and M2951 respectively) and cloned into 449 pEFGPT12S-CvectorEGFPmyc/XmaI/SacII to produce pCVecGFP184 (GFP at the N-450 terminus). Mutations were introduced into the FPV184 gene by PCR using the 451 following primer pairs (as shown in Fig. 5c Following transfection of constructs into FP9-infected CEF, recombinant viruses were 457 selected for, using mycophenolic acid, and plaque purified twice. 458

Cloning for ectopic expression of viral genes 459
The

Infection of CEF for microarray and qPCR analyses 466
Media was removed from fully confluent CEF (in T25 flasks; Greiner Bio One; 5.6 x 467  Table S1]. 514

Quantitative real-time RT PCR 515
Quantitative real-time RT PCR was performed on RNA samples using a two-step 516 procedure as previously 50 . RNA was first reverse-transcribed into cDNA using the 517 QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer's 518 instructions. qPCR was then conducted on the cDNA in a 384-well plate with an ABI-519 7900HT Fast qPCR system (Applied Biosystems). Mesa Green qPCR MasterMix 520 (Eurogentec) was added to the cDNA (5 μl for every 2 μl of cDNA). The following 521 amplification conditions were used: 95°C for 5 min; 40 cycles of 95°C for 15 sec, 57°C 522 for 20 sec, and 72°C for 20 sec; 95°C for 15 sec; 60°C for 15 sec; and 95°C for 15 523 sec. Primer sequences for genes that were used in the study are shown in S6. The 524 output Ct values and dissociation curves were analysed using SDS v2.3 and RQ 525 Manager v1.2 (Applied Biosystems). Gene expression data were normalized against 526 the housekeeping gene GAPDH, and compared with the mock controls using the 527 comparative CT method [also referred to as the 2 -ΔΔCT method 51 ]. All samples were 528 loaded in triplicate. 529 530

Confocal and widefield fluorescence microscopy 531
Immunofluorescence labelling was carried out using CEF seeded at 2.5 x 10 5 cells/well 532 on coverslips, incubated in 6 well plates at 37°C, 5% CO2 and infected with 0.5 -1 pfu 533 of virus for 24 h. Medium was aspirated, cells washed 3x with PBS and fixed with 4% 534 paraformaldehyde in PBS at room temperature (RT). Coverslips were washed 3x in 535 PBS and cells permeabilised (0.5% Triton X-100 in PBS) at RT for 10 min with shaking. 536 The amino acid sequences of FPV184 orthologues from each genera of 641 chordopoxviruses were subjected to multiple alignments (Fig. 5a

Statistical analysis 652
To determine the significance of differences between experimental groups, one-or 653 two-way ANOVA analyses were performed using the fold change scores with a 654