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Lyssavirus P Protein Isoforms Diverge Significantly in Subcellular Interactions Underlying Mechanisms of Interferon Antagonism

October 2022
96(20):e0139622

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Viral hijacking of microtubule (MT)-dependent transport is well understood, but several viruses also express discrete MT-associated proteins (vMAPs), potentially to modulate MT-dependent processes in the host cell. Specific roles for vMAP-MT interactions include subversion of antiviral responses by P3, an isoform of the P protein of rabies virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon (IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3 also undergoes nucleocytoplasmic trafficking and inhibits STAT1-DNA binding, indicative of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antagonist functions of P3 correlate with pathogenesis, indicating potential as therapeutic targets. However, key questions remain, including whether other P protein isoforms interact with MTs, the relationship of these interactions with pathogenesis, and the extent of conservation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-resolution microscopy, live-cell imaging, and immune signaling analyses, we find that multiple P protein isoforms associate with MTs and that association correlates with pathogenesis. Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular localization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within both phylogroup I and II. Nevertheless, the divergent P3 proteins retain significant IFN antagonist function, indicative of adaptation to favor different inhibitory mechanisms, with MT interaction important to phylogroup I viruses. IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year. Viral subversion of host cell functions, including antiviral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to the virus-host interface underpinning immune evasion. Here, we show that specific cellular interactions of P protein isoforms involved in immune evasion vary significantly between different lyssaviruses, indicative of distinct strategies to evade immune responses. These findings highlight the diversity of the virus-host interface, an important consideration in the development of pan-lyssavirus therapeutic approaches.

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Lyssavirus P Protein Isoforms Diverge Signiﬁcantly in

Subcellular Interactions Underlying Mechanisms of Interferon

Antagonism

Aaron M. Brice,a,b Ashley M. Rozario,cStephen M. Rawlinson,a,b Cassandra T. David,a,b Linda Wiltzer-Bach,bDavid A. Jans,dNaoto Ito,e,f

Toby D. M. Bell,cGregory W. Moseleya,b

Viral Pathogenesis Laboratory, Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia

Viral Pathogenesis Laboratory, Department of Microbiology, Monash University, Clayton, Victoria, Australia

School of Chemistry, Monash University, Clayton, Victoria, Australia

Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan

United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan

ABSTRACT Viral hijacking of microtubule (MT)-dependent transport is well understood,

but several viruses also express discrete MT-associated proteins (vMAPs), potentially to

modulate MT-dependent processes in the host cell. Speciﬁc roles for vMAP-MT interac-

tions include subversion of antiviral responses by P3, an isoform of the P protein of rabies

virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon

(IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3

also undergoes nucleocytoplasmic trafﬁcking and inhibits STAT1-DNA binding, indicative

of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antag-

onist functions of P3 correlate with pathogenesis, indicating potential as therapeutic tar-

gets. However, key questions remain, including whether other P protein isoforms interact

with MTs, the relationship of these interactions with pathogenesis, and the extent of con-

servation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-reso-

lution microscopy, live-cell imaging, and immune signaling analyses, we ﬁnd that multiple

P protein isoforms associate with MTs and that association correlates with pathogenesis.

Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular local-

ization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT

association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while

nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within

both phylogroup I and II. Nevertheless, the divergent P3 proteins retain signiﬁcant IFN an-

tagonist function, indicative of adaptation to favor different inhibitory mechanisms, with

MT interaction important to phylogroup I viruses.

IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomy-

elitis that is almost invariably fatal. There are no effective antivirals for symptomatic infec-

tion, and effective application of current vaccines is limited in areas of endemicity, such that

rabies causes ;59,000 deaths per year. Viral subversion of host cell functions, including anti-

viral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to

the virus-host interface underpinning immune evasion. Here, we show that speciﬁc cellular

interactions of P protein isoforms involved in immune evasion vary signiﬁcantly between

different lyssaviruses, indicative of distinct strategies to evade immune responses. These

ﬁndings highlight the diversity of the virus-host interface, an important consideration in the

development of pan-lyssavirus therapeutic approaches.

KEYWORDS immune evasion, lyssavirus, microtubules, nuclear localisation, rabies

virus, super-resolution microscopy

Editor Susana López, Instituto de

Biotecnologia/UNAM

Address correspondence to Gregory W.

Moseley, greg.moseley@monash.edu.

The authors declare no conﬂict of interest.

Received 9 September 2022

Accepted 9 September 2022

Published 12 October 2022

October 2022 Volume 96 Issue 20 10.1128/jvi.01396-22 1

VIRUS-CELL INTERACTIONS

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The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

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To evade immunity, many viruses express interferon antagonists that target STAT transcription factors as a major component of pathogenesis. Because of a lack of direct structural data, these interfaces are poorly understood. We report the structural analysis of full-length STAT1 binding to an interferon antagonist of a human pathogenic virus. The interface revealed by transferred cross-saturation NMR is complex, involving multiple regions in both the viral and cellular proteins. Molecular mapping analysis, combined with biophysical characterization and in vitro/in vivo functional assays, indicates that the interface is significant in disease caused by a pathogenic field-strain lyssavirus, with critical roles for contacts between the STAT1 coiled-coil/DNA-binding domains and specific regions within the viral protein. These data elucidate the potentially complex nature of IFN antagonist/STAT interactions, and the spatial relationship of protein interfaces that mediate immune evasion and replication, providing insight into how viruses can regulate these essential functions via single multifunctional proteins.

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Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and PML nuclear bodies, and interacts with nucleolin and PML, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localisation of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni), and non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

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Lyssavirus P Protein Isoforms Diverge Significantly in Subcellular Interactions Underlying Mechanisms of Interferon Antagonism

Abstract

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