Differential Innate Immune Signaling in Macrophages by Wild-Type Vaccinia Mature Virus and a Mutant Virus with a Deletion of the A26 Protein

Abstract

The Western Reserve (WR) strain of mature vaccinia virus contains an A26 envelope protein that mediates virus binding to cell surface laminin and subsequent endocytic entry into HeLa cells. Removal of the A26 protein from the WR strain mature virus generates a mutant, WRΔA26, that enters HeLa cells through plasma membrane fusion. Here, we infected murine bone marrow-derived macrophages (BMDM) with wild-type strain WR and the WRΔA26 mutant and analyzed viral gene expression and cellular innate immune signaling. In contrast to previous studies, in which both HeLa cells infected with WR and HeLa cells infected with WRΔA26 expressed abundant viral late proteins, we found that WR expressed much less viral late protein than WRΔA26 in BMDM. Microarray analysis of the cellular transcripts in BMDM induced by virus infection revealed that WR preferentially activated type 1 interferon receptor (IFNAR)-dependent signaling but WRΔA26 did not. We consistently detected a higher level of soluble beta interferon secretion and phosphorylation of the STAT1 protein in BMDM infected with WR than in BMDM infected with WRΔA26. When IFNAR-knockout BMDM were infected with WR, late viral protein expression increased, confirming that IFNAR-dependent signaling was differentially induced by WR and, in turn, restricted viral late gene expression. Finally, wild-type C57BL/6 mice were more susceptible to mortality from WRΔA26 infection than to that from WR infection, whereas IFNAR-knockout mice were equally susceptible to WR and WRΔA26 infection, demonstrating that the ability of WRΔA26 to evade IFNAR signaling has an important influence on viral pathogenesis The vaccinia virus A26 protein was previously shown to mediate virus attachment and to regulate viral endocytosis. Here, we show that infection with strain WR induces a robust innate immune response that activates type 1 interferon receptor (IFNAR)-dependent cellular genes in BMDM, whereas infection with the WRΔA26 mutant does not. We further demonstrated that the differential activation of IFNAR-dependent cellular signaling between WR and WRΔA26 not only is important for differential host restriction in BMDM but also is important for viral virulence Our study reveals a new property of WRΔA26, which is in regulating host antiviral innate immunity and.

Vaccinia viral A26 protein is a fusion suppressor of mature virus and triggers membrane fusion through conformational change at low pH

“…Recombinant viruses expressing WR-A26(76–500) or WR-A26(321–500) were isolated and plaque-purified. A recombinant vaccinia virus expressing full-length flag-tagged A26 protein, named WR-A26, was also included [36]. HeLa cells were infected with individual virus at a multiplicity of infection (MOI) of 5 plaque-forming units (PFU) per cell and harvested at 24 hours post infection (hpi) to determine MV growth (Fig 1C).…”

“…Schematics of recombinant viruses showing the WR-A26-H2R and WR-A26-H3R mutant genome arrangements. WR-A26 and WR-ΔA26 were described previously [32, 36], and the latter was used as the parental virus to generate the WR-A26-H2R and WR-A26-H3R recombinant viruses. The A26-H2R and A26-H3R gene cassettes were inserted into the A26L native locus and recombinant virus was selected as blue plaques in agar plates containing X-gal.…”

“…Another advantage of having multiple entry pathways is to avoid innate immune sensing and antiviral signaling activation. We recently infected murine bone marrow-derived macrophages (BMDM) with WR or WR-ΔA26 virus and found that the IFNβ-Stat1 signaling pathway was preferentially induced by endocytic WR virus but not by WR-ΔA26 virus [36]. Consequently, WR-ΔA26 exhibited enhanced virulence in mice compared to WR vaccinia virus [36].…”

Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56/K2 Protein Complex

“…Viral genomic DNA containing an early Venus expression cassette flanked by J4L and J5R sequences was obtained from John H. Connor (54,55) and used as the template to obtain a PCR fragment, which was subsequently cloned into a TOPO plasmid, resulting in pTopo-J4R-Venus-J5L, in which Venus is expressed from a viral early promoter of the C11R gene (54). The plasmid was sequenced to ensure accuracy and subsequently transfected into CV-1 cells that had been infected with WRΔA26-A4mCherry (50). The lysates were harvested at 24 h postinfection (hpi), and virus titration was performed on BSC40 cells to isolate the WRΔA26-Venus-A4-mCherry virus through multiple rounds of plaque purification by fluorescence microscopy until 100% purity was reached.…”

Modulating Vaccinia Virus Immunomodulators to Improve Immunological Memory

“…Many other VACV proteins, either immunomodulators or proteins with other functions, affect virulence and/or the host immune response. These include I4 (ribonucleotide reductase large subunit) [ 271 , 272 ], J2 (thymidine kinase) [ 273 ], A26 (virion attachment/entry) [ 274 ], B5 (virion envelope glycoprotein related to complement control factor) [ 275 , 276 ], B13 and B22 (serine protease inhibitors SPI-2 and SPI-1, respectively) [ 163 , 277 , 278 , 279 ], F12 (IEV transport protein) [ 280 ], A36 (IEV transport protein and actin tail inducer) [ 163 , 281 ], A40 (cell surface glycoprotein) [ 163 , 282 ], A50 (DNA ligase) [ 283 ], C2, F3 and A55 (intracellular kelch protein immunomodulators) [ 284 , 285 , 286 ] and A33 (virion envelope glycoprotein) [ 276 ], However, the protective capacity of viruses lacking these proteins requires further study.…”