Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4

Jesus, Desyreé Murta; Moussatché, Nissin; McFadden, Baron B.D.; Nielsen, Casey P.; D’Costa, Susan; Condit, Richard C.

doi:10.1016/j.virol.2015.02.020

Cited by 11 publications

(7 citation statements)

References 33 publications

(51 reference statements)

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“…2A). In addition, iF17 produced lower levels of the late structural protein A10, which was also notably not processed to its faster-migrating form, which occurs during virion maturation (81). This is in line with prior reports of late-stage defects in virion maturation in the absence of F17, which involve failed processing of other structural proteins (46,47), and suggests that, although this mutant proceeds through early stages of infection, it subsequently encounters both postreplicative-protein production and virion maturation defects.…”

Section: Resultsmentioning

confidence: 95%

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

Meade

King

Munger

et al. 2019

J Virol

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Despite producing enormous amounts of cytoplasmic DNA, poxviruses continue to replicate efficiently by deploying an armory of proteins that counter host antiviral responses at multiple levels. Among these, poxvirus protein F17 dysregulates the host kinase mammalian target of rapamycin (mTOR) to prevent the activation of stimulator of interferon genes (STING) expression and impair the production of interferon-stimulated genes (ISGs). However, the host DNA sensor(s) involved and their impact on infection in the absence of F17 remain unknown. Here, we show that cyclic-di-GMP-AMP (cGAMP) synthase (cGAS) is the primary sensor that mediates interferon response factor (IRF) activation and ISG responses to vaccinia virus lacking F17 in both macrophages and lung fibroblasts, although additional sensors also operate in the latter cell type. Despite this, ablation of ISG responses through cGAS or STING knockout did not rescue defects in late-viral-protein production, and the experimental data pointed to other functions of mTOR in this regard. mTOR adjusts both autophagic and protein-synthetic processes to cellular demands. No significant differences in autophagic responses to wild-type or F17 mutant viruses could be detected, with autophagic activity differing across cell types or states and exhibiting no correlations with defects in viral-protein accumulation. In contrast, results using transformed cells or altered growth conditions suggested that late-stage defects in protein accumulation reflect failure of the F17 mutant to deregulate mTOR and stimulate protein production. Finally, rescue approaches suggest that phosphorylation may partition F17’s functions as a structural protein and mTOR regulator. Our findings reveal the complex multifunctionality of F17 during infection. IMPORTANCE Poxviruses are large, double-stranded DNA viruses that replicate entirely in the cytoplasm, an unusual act that activates pathogen sensors and innate antiviral responses. In order to replicate, poxviruses therefore encode a wide range of innate immune antagonists that include F17, a protein that dysregulates the kinase mammalian target of rapamycin (mTOR) to suppress interferon-stimulated gene (ISG) responses. However, the host sensor(s) that detects infection in the absence of F17 and its precise contribution to infection remains unknown. Here, we show that the cytosolic DNA sensor cGAS is primarily responsible for activating ISG responses in biologically relevant cell types infected with a poxvirus that does not express F17. However, in line with their expression of ∼100 proteins that act as immune response and ISG antagonists, while F17 helps suppress cGAS-mediated responses, we find that a critical function of its mTOR dysregulation activity is to enhance poxvirus protein production.

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Section: Resultsmentioning

confidence: 95%

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

Meade

King

Munger

et al. 2019

J Virol

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“…We speculate that A12 may span the core wall with fragment 3 contacting transcriptosome and p4b interiorly, and fragments 1 and 2 oriented towards the exterior contacting A4, p4a, F17 and TM proteins H3, F9 and H2 (S1 Fig). The compromised VP8 encapsidation upon repression of core wall protein p4b [48] seems consistent with a core wall connection to VP8 also, as does the compromised core wall in absence of VP8 [49]. Since, minimally, ~57 amino acids (aa) of linear beta sheet would be required cross a 20 nm core wall, contacts within A12 of the C-terminal 20% of VP8 and some membrane proteins, appear to be more intimate than a wall’s-width.…”

Section: Resultsmentioning

confidence: 99%

The Vaccinia virion: Filling the gap between atomic and ultrastructure

Mirzakhanyan

Gershon

2019

PLoS Pathog

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We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an ‘attachment protein’ sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.

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“…The function of A3 has been studied previously using temperature-sensitive mutants where it was found that virions formed at non-permissive temperatures were abnormal in shape, had substantially reduced infectivity, and reached transcription levels less than 2% of the wild-type virus, thus indicating A3 plays a critical role in virion morphogenesis [44]. Additionally, an inducible A3L virus has been generated using lac operon elements in a system where the bacteriophage T7 RNA polymerase is expressed under a late promoter controlled by a lac operator, and the A3L gene is placed under a T7 promoter also controlled by a lac operator [64]. In the presence of inducer (IPTG) the virus formed significantly smaller plaques, and in the absence of inducer, abnormal immature virions accumulated within the cytoplasm and no plaques were observed, demonstrating that A3 is required for normal virion formation.…”

Section: Plos Onementioning

confidence: 99%

Replication-inducible vaccinia virus vectors with enhanced safety in vivo

et al. 2020

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Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replicationcompetent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of tet operon elements to regulate their expression with tetracycline antibiotics. These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety.

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Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4

Cited by 11 publications

References 33 publications

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

The Vaccinia virion: Filling the gap between atomic and ultrastructure

Replication-inducible vaccinia virus vectors with enhanced safety in vivo

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