Publications by authors named "Justin A Roby"

22 Publications

  • Page 1 of 1

Correction: Tsimbalyuk et al. The Intrinsically Disordered W Protein Is Multifunctional during Henipavirus Infection, Disrupting Host Signalling Pathways and Nuclear Import. , , 1913.

Cells 2022 Apr 11;11(8). Epub 2022 Apr 11.

School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.

The authors would like to make the following corrections to the published paper [...].
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http://dx.doi.org/10.3390/cells11081290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9027788PMC
April 2022

MERS-CoV ORF4b employs an unusual binding mechanism to target IMPα and block innate immunity.

Nat Commun 2022 03 25;13(1):1604. Epub 2022 Mar 25.

School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia.

The MERS coronavirus (MERS-CoV) is a highly pathogenic, emerging virus that produces accessory proteins to antagonize the host innate immune response. The MERS-CoV ORF4b protein has been shown to bind preferentially to the nuclear import adapter IMPα3 in infected cells, thereby inhibiting NF-κB-dependent innate immune responses. Here, we report high-resolution structures of ORF4b bound to two distinct IMPα family members. Each exhibit highly similar binding mechanisms that, in both cases, lack a prototypical Lys bound at their P2 site. Mutations within the NLS region dramatically alter the mechanism of binding, which reverts to the canonical P2 Lys binding mechanism. Mutational studies confirm that the novel binding mechanism is important for its nuclear import, IMPα interaction, and inhibition of innate immune signaling pathways. In parallel, we determined structures of the nuclear binding domain of NF-κB component p50 bound to both IMPα2 and α3, demonstrating that p50 overlaps with the ORF4b binding sites, suggesting a basis for inhibition. Our results provide a detailed structural basis that explains how a virus can target the IMPα nuclear import adapter to impair immunity, and illustrate how small mutations in ORF4b, like those found in closely related coronaviruses such as HKU5, change the IMPα binding mechanism.
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http://dx.doi.org/10.1038/s41467-022-28851-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8956657PMC
March 2022

Structural characterization of the porcine adeno-associated virus Po1 capsid protein binding to the nuclear trafficking protein importin alpha.

FEBS Lett 2021 11 1;595(22):2793-2804. Epub 2021 Nov 1.

School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.

Adeno-associated viruses (AAVs) are key vectors for gene therapy; thus, many aspects of their cell transduction pathway have been revealed in detail. However, the specific mechanisms AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the structural interactions between the AAV capsid (Cap) protein and the nuclear transport protein importin alpha (IMPα). A putative nuclear localization sequence (NLS) in the virion protein 1 capsid protein of the porcine AAV Po1 was identified. This region was complexed with IMPα and a structure solved at 2.26 Å. This is the first time that an NLS of AAV Cap complexed with IMPα has been determined structurally. Our results support the findings that AAV capsids enter the nucleus through binding the nuclear import adapter IMPα.
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http://dx.doi.org/10.1002/1873-3468.14209DOI Listing
November 2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity.

Elife 2021 08 3;10. Epub 2021 Aug 3.

Department of Immunology, School of Medicine, University of Washington, Seattle, United States.

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.
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http://dx.doi.org/10.7554/eLife.71047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357416PMC
August 2021

A scoping review of African swine fever virus spread between domestic and free-living pigs.

Transbound Emerg Dis 2021 Sep 7;68(5):2643-2656. Epub 2021 Feb 7.

School of Animal and Veterinary Sciences, Faculty of Science, Charles Sturt University, Wagga Wagga, NSW, Australia.

Since 2007, African swine fever virus (ASFV) has spread to countries in Europe, Asia and Oceania and has caused devastating impacts on pigs and the pork industry. Transmission can be direct or indirect, and epidemiologic scenarios have been described in which spread occurs between free-living and domestic pigs. The purpose of this scoping review was to identify primary research in which authors made statements to support ASFV transmission between free-living and domestic pigs and assess the circumstances in which transmission events occurred. A search was conducted in five bibliographic databases and the grey literature. Two reviewers (from a team of ten) independently screened each record and charted data (demographics of the pig populations, their husbandry [domestic pigs] and habitat [free-living pigs], the spatial and temporal distribution of ASF, the occurrence or burden of ASF in the populations, and whether ticks were present in the geographic range of the pig populations). Data synthesis included statistics and a narrative summary. From 1,349 records screened, data were charted from 46 individual studies published from 1985 to 2020. Outbreak investigations revealed that whilst poor biosecurity of domestic pig operations was often reported, direct contact resulting in transmission between free-living and domestic pigs was rarely reported. Studies in which quantitative associations were made generally found that spread within populations was more important than spread between populations, although this was not always the case, particularly when domestic pigs were free-ranging. We conclude that there is limited evidence that transmission of ASFV between free-living and domestic pigs is an important feature of ASF epidemiology, especially in the current ASF epidemic in Europe and the Russian Federation. If ASFV elimination cannot be achieved in free-living pigs, compartmentalization of domestic pig populations from free-living populations via biosecurity strategies could be used to support trade of domestic pigs.
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http://dx.doi.org/10.1111/tbed.13993DOI Listing
September 2021

Structural Perspectives of Beak and Feather Disease Virus and Porcine Circovirus Proteins.

Viral Immunol 2021 Jan-Feb;34(1):49-59. Epub 2020 Dec 4.

School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia.

Circoviruses represent a rapidly expanding group of viruses that infect both vertebrate and invertebrate hosts. Members are responsible for diseases of veterinary and economic importance, including postweaning multisystemic wasting syndrome in pigs, and beak and feather disease (BFD) in birds. These viruses are associated with lymphoid depletion and immunosuppressive conditions in infected animals leading to systemic illness. Circoviruses are small nonenveloped DNA viruses containing a single-stranded circular genome, encoding two major proteins: the capsid-associated protein (Cap), comprising the entirety of the viral capsid, and the replication-associated protein (Rep). Cap is the only protein component of the virion and plays crucial roles throughout the virus replication cycle, including viral attachment, cell entry, genome uncoating, and packaging of newly formed viral particles. Rep mediates recognition of replication origin motifs in the viral genome sequence and is responsible for endonuclease activity enabling nicking of the circular DNA and initiation of rolling-circle replication (RCR). Porcine circovirus 2 (PCV2) was the first circovirus capsid structure to be solved at atomic resolution using X-ray crystallography. The structure revealed an assembly comprising 60 monomeric subunits to form virus-like particles. Each Cap monomer harbors a canonical viral jelly roll domain composed of two, four-stranded antiparallel -sheets. Crystal structures of two distinct macromolecular assemblies from BFD virus Cap were also resolved at high resolution. In these structures, the exposure of the N-terminal arginine-rich motif, responsible for DNA binding and nuclear localization is reversed. Additional structural investigations have also elucidated a PCV2 type-specific neutralizing epitope, and interaction between the PCV2 capsid and polymers such as heparin. In this review, we provide a snapshot of the structural and functional aspects of circovirus proteins.
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http://dx.doi.org/10.1089/vim.2020.0097DOI Listing
February 2022

The Structural Features of Matrix Protein Driving Intracellular Trafficking.

Viral Immunol 2021 Jan-Feb;34(1):27-40. Epub 2020 Oct 6.

School of Biomedical Sciences and Charles Sturt University, Wagga Wagga, Australia.

Henipaviruses are single-stranded RNA viruses that have recently emerged as zoonotic pathogens, capable of causing severe acute respiratory disease and encephalitis in humans. The prototypical henipaviruses, and , are a major health concern as they have high mortality rates and no currently approved human vaccine or drug therapy. Understanding the mechanisms of viral replication and pathogenicity is of critical importance for therapeutic developments. A novel target for such therapies is the Matrix (M) protein, a multifunctional protein that drives viral assembly and inhibits the innate immune response. These multifunctional attributes promote a complicated lifecycle: while viral replication occurs in the cytoplasm, M traffics to the nucleus, where it is ubiquitinated, for correct cellular targeting and virion packaging. In this study, we review the relationship between the structure and functions of M. In specific cases, the compatibility between structural accessibility and protein functionality is not always evident, and we highlight areas that require further investigation.
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http://dx.doi.org/10.1089/vim.2020.0056DOI Listing
February 2022

The Intrinsically Disordered W Protein Is Multifunctional during Henipavirus Infection, Disrupting Host Signalling Pathways and Nuclear Import.

Cells 2020 08 18;9(8). Epub 2020 Aug 18.

School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.

Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host's innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics.
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http://dx.doi.org/10.3390/cells9081913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465373PMC
August 2020

Proteome-Wide Zika Virus CD4 T Cell Epitope and HLA Restriction Determination.

Immunohorizons 2020 08 4;4(8):444-453. Epub 2020 Aug 4.

Department of Medicine, University of Washington, Seattle, WA 98195;

Zika virus (ZIKV) is a mosquito-borne pathogen that caused an epidemic in 2015-2016. ZIKV-specific T cell responses are functional in animal infection models, and helper CD4 T cells promote avid Abs in the vaccine context. The small volumes of blood available from field research limit the determination of T cell epitopes for complex microbes such as ZIKV. The goal of this project was efficient determination of human ZIKV CD4 T cell epitopes at the whole proteome scale, including validation of reactivity to whole pathogen, using small blood samples from convalescent time points when T cell response magnitude may have waned. Polyclonal enrichment of candidate ZIKV-specific CD4 T cells used cell-associated virus, documenting that T cells in downstream peptide analyses also recognize whole virus after Ag processing. Sequential query of bulk ZIKV-reactive CD4 T cells with pooled/single ZIKV peptides and molecularly defined APC allowed precision epitope and HLA restriction assignments across the ZIKV proteome and enabled discovery of numerous novel ZIKV CD4 T cell epitopes. The research workflow is useful for the study of emerging infectious diseases with a very limited human blood sample availability.
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http://dx.doi.org/10.4049/immunohorizons.2000068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839664PMC
August 2020

Flavivirus Nonstructural Protein NS5 Dysregulates HSP90 to Broadly Inhibit JAK/STAT Signaling.

Cells 2020 04 7;9(4). Epub 2020 Apr 7.

Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109, USA.

Pathogenic flaviviruses antagonize host cell Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling downstream of interferons α/β. Here, we show that flaviviruses inhibit JAK/STAT signaling induced by a wide range of cytokines beyond interferon, including interleukins. This broad inhibition was mapped to viral nonstructural protein 5 (NS5) binding to cellular heat shock protein 90 (HSP90), resulting in reduced Janus kinase-HSP90 interaction and thus destabilization of unchaperoned JAKs (and other kinase clients) of HSP90 during infection by , West Nile virus, and Japanese encephalitis virus. Our studies implicate viral dysregulation of HSP90 and the JAK/STAT pathway as a critical determinant of cytokine signaling control during flavivirus infection.
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http://dx.doi.org/10.3390/cells9040899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226784PMC
April 2020

RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions.

Nat Immunol 2019 12 18;20(12):1610-1620. Epub 2019 Nov 18.

Department of Immunology, School of Medicine, University of Washington, Seattle, WA, USA.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.
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http://dx.doi.org/10.1038/s41590-019-0527-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240801PMC
December 2019

Immune Evasion Strategies Used by Zika Virus to Infect the Fetal Eye and Brain.

Viral Immunol 2020 Jan/Feb;33(1):22-37. Epub 2019 Nov 5.

Center for Innate Immunity and Immune Disease, University of Washington, Seattle, Washington.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused a public health emergency in the Americas when an outbreak in Brazil became linked to congenital microcephaly. Understanding how ZIKV could evade the innate immune defenses of the mother, placenta, and fetus has become central to determining how the virus can traffic into the fetal brain. ZIKV, like other flaviviruses, evades host innate immune responses by leveraging viral proteins and other processes that occur during viral replication to allow spread to the placenta. Within the placenta, there are diverse cell types with coreceptors for ZIKV entry, creating an opportunity for the virus to establish a reservoir for replication and infect the fetus. The fetal brain is vulnerable to ZIKV, particularly during the first trimester, when it is beginning a dynamic process, to form highly complex and specialized regions orchestrated by neuroprogenitor cells. In this review, we provide a conceptual framework to understand the different routes for viral trafficking into the fetal brain and the eye, which are most likely to occur early and later in pregnancy. Based on the injury profile in human and nonhuman primates, ZIKV entry into the fetal brain likely occurs across both the blood/cerebrospinal fluid barrier in the choroid plexus and the blood/brain barrier. ZIKV can also enter the eye by trafficking across the blood/retinal barrier. Ultimately, the efficient escape of innate immune defenses by ZIKV is a key factor leading to viral infection. However, the host immune response against ZIKV can lead to injury and perturbations in developmental programs that drive cellular division, migration, and brain growth. The combined effect of innate immune evasion to facilitate viral propagation and the maternal/placental/fetal immune response to control the infection will determine the extent to which ZIKV can injure the fetal brain.
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http://dx.doi.org/10.1089/vim.2019.0082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978768PMC
February 2021

Comparative Analysis of African and Asian Lineage-Derived Zika Virus Strains Reveals Differences in Activation of and Sensitivity to Antiviral Innate Immunity.

J Virol 2019 07 14;93(13). Epub 2019 Jun 14.

Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA

In recent years, Asian lineage Zika virus (ZIKV) strains emerged to cause pandemic outbreaks associated with a high rate of congenital ZIKV syndrome (CZVS). The reasons for the enhanced spread and severe disease caused by newly emerging strains are not fully understood. Here we compared viral sequences, viral replication, and innate immune signaling induction of three different ZIKV strains derived from African and Asian lineages and West Nile virus, another flavivirus. We found pronounced differences in activation of innate immune signaling and inhibition of viral replication across ZIKV strains. The newly emerged Asian ZIKV strain Brazil Fortaleza 2015, which is associated with a higher rate of neurodevelopmental disorders like microcephaly, induced much weaker and delayed innate immune signaling in infected cells. However, superinfection studies to assess control of innate immune signaling induced by Sendai virus argue against an active block of IRF3 activation by the Brazilian strain of ZIKV and rather suggest an evasion of detection by host cell pattern recognition receptors. Compared to the Asian strain FSS13025 isolated in Cambodia, both ZIKV Uganda MR766 and ZIKV Brazil Fortaleza appear less sensitive to the interferon-induced antiviral response. ZIKV infection studies of cells lacking the different RIG-I-like receptors identified RIG-I as the major cytosolic pattern recognition receptor for detection of ZIKV. Zika Virus (ZIKV), discovered in 1947, is divided into African and Asian lineages. Pandemic outbreaks caused by currently emerging Asian lineage strains are accompanied by high rates of neurological disorders and exemplify the global health burden associated with this virus. Here we compared virological and innate immunological aspects of two ZIKV strains from the Asian lineage, an emerging Brazilian strain and a less-pathogenic Cambodian strain, and the prototypic African lineage ZIKV strain from Uganda. Compared to the replication of other ZIKV strains, the replication of ZIKV Brazil was less sensitive to the antiviral actions of interferon (IFN), while infection with this strain induced weaker and delayed innate immune responses Our data suggest that ZIKV Brazil directs a passive strategy of innate immune evasion that is reminiscent of a stealth virus. Such strain-specific properties likely contribute to differential pathogenesis and should be taken into consideration when choosing virus strains for future molecular studies.
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http://dx.doi.org/10.1128/JVI.00640-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580957PMC
July 2019

Interleukin-1β Induces mtDNA Release to Activate Innate Immune Signaling via cGAS-STING.

Mol Cell 2019 05 2;74(4):801-815.e6. Epub 2019 Apr 2.

Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109, USA. Electronic address:

Interleukin-1 beta (IL-1β) is a pleiotropic mediator of inflammation and is produced in response to a wide range of stimuli. During infection, IL-1β production occurs in parallel with the onset of innate antimicrobial defenses, but the contribution of IL-1β signaling to cell-intrinsic immunity is not defined. Here, we report that exogenous IL-1β induces interferon regulatory factor 3 (IRF3) activation in human myeloid, fibroblast, and epithelial cells. IRF3 activation by IL-1β is dependent upon the DNA-sensing pathway adaptor, stimulator of interferon genes (STING), through the recognition of cytosolic mtDNA by cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). IL-1β treatment results in interferon (IFN) production and activation of IFN signaling to direct a potent innate immune response that restricts dengue virus infection. This study identifies a new function for IL-1β in the onset or enhancement of cell-intrinsic immunity, with important implications for cGAS-STING in integrating inflammatory and microbial cues for host defense.
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http://dx.doi.org/10.1016/j.molcel.2019.02.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596306PMC
May 2019

Zika virus and the nonmicrocephalic fetus: why we should still worry.

Am J Obstet Gynecol 2019 01 29;220(1):45-56. Epub 2018 Aug 29.

Department of Obstetrics and Gynecology and Global Health, University of Washington, Seattle, WA; Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA; Sahlgrenska Academy, Gothenburg University, Gothenburg Sweden. Electronic address:

Zika virus is a mosquito-transmitted flavivirus and was first linked to congenital microcephaly caused by a large outbreak in northeastern Brazil. Although the Zika virus epidemic is now in decline, pregnancies in large parts of the Americas remain at risk because of ongoing transmission and the potential for new outbreaks. This review presents why Zika virus is still a complex and worrisome public health problem with an expanding spectrum of birth defects and how Zika virus and related viruses evade the immune response to injure the fetus. Recent reports indicate that the spectrum of fetal brain and other anomalies associated with Zika virus exposure is broader and more complex than microcephaly alone and includes subtle fetal brain and ocular injuries; thus, the ability to prenatally diagnose fetal injury associated with Zika virus infection remains limited. New studies indicate that Zika virus imparts disproportionate effects on fetal growth with an unusual femur-sparing profile, potentially providing a new approach to identify viral injury to the fetus. Studies to determine the limitations of prenatal and postnatal testing for detection of Zika virus-associated birth defects and long-term neurocognitive deficits are needed to better guide women with a possible infectious exposure. It is also imperative that we investigate why the Zika virus is so adept at infecting the placenta and the fetal brain to better predict other viruses with similar capabilities that may give rise to new epidemics. The efficiency with which the Zika virus evades the early immune response to enable infection of the mother, placenta, and fetus is likely critical for understanding why the infection may either be fulminant or limited. Furthermore, studies suggest that several emerging and related viruses may also cause birth defects, including West Nile virus, which is endemic in many parts of the United States. With mosquito-borne diseases increasing worldwide, there remains an urgent need to better understand the pathogenesis of the Zika virus and related viruses to protect pregnancies and child health.
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http://dx.doi.org/10.1016/j.ajog.2018.08.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501788PMC
January 2019

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain.

Nat Med 2018 03 5;24(3):368-374. Epub 2018 Feb 5.

Washington National Primate Research Center, Seattle, Washington, USA.

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth.
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http://dx.doi.org/10.1038/nm.4485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839998PMC
March 2018

Rapid reversal of innate immune dysregulation in blood of patients and livers of humanized mice with HCV following DAA therapy.

PLoS One 2017 17;12(10):e0186213. Epub 2017 Oct 17.

Division of Gastroenterology and Hepatology, Hepatitis C Center, Department of Medicine, University of Colorado Denver (UCD), Aurora, Colorado, United States of America.

Results: First, in patients receiving two different combinations of DAAs, we found that DAAs induced not only rapid viral clearance, but also a re-setting of antiviral immune responses in the peripheral blood. Specifically, we see a rapid decline in the expression of genes associated with chronic IFN stimulation (IFIT3, USP18, IFIT1) as well as a rapid decline in genes associated with inflammation (IL1β, CXCL10, CXCL11) in the peripheral blood that precedes the complete removal of virus from the blood. Interestingly, this rapid reversal of innate immune activation was not seen in patients who successfully clear chronic HCV infection using IFN-based therapy. Next, using a novel humanized mouse model (Fah-/-RAG2-/-IL2rgnull-FRG), we assessed the changes that occur in the hepatic tissue following DAA treatment. DAA-mediated rapid HCV clearance resulted in blunting of the expression of proinflammatory responses while functionally restoring the RIG-I/MAVS axis in the liver of humanized mice.

Conclusions: Collectively, our data demonstrate that the rapid viral clearance following treatment with DAAs results in the rebalancing of innate antiviral response in both the peripheral blood and the liver as well as enhanced antiviral signaling within previously infected hepatocytes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186213PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645093PMC
October 2017

Post-translational regulation and modifications of flavivirus structural proteins.

J Gen Virol 2015 Jul 23;96(Pt 7):1551-69. Epub 2015 Feb 23.

1Australian Infectious Diseases Research Centre, The University of Queensland, Australia 2School of Chemistry and Molecular Biosciences, The University of Queensland, Australia.

Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.
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http://dx.doi.org/10.1099/vir.0.000097DOI Listing
July 2015

Increased expression of capsid protein in trans enhances production of single-round infectious particles by West Nile virus DNA vaccine candidate.

J Gen Virol 2014 Oct 23;95(Pt 10):2176-2191. Epub 2014 Jun 23.

School of Chemistry and Molecular Biosciences, University of Queensland, Australia.

West Nile virus (WNV; genus Flavivirus, family Flaviviridae) is an emerging pathogenic arbovirus responsible for outbreaks of encephalitis around the world. Whilst no vaccines are currently available to prevent WNV infection of humans, the use of cDNA copies of flavivirus RNA genomes with large internal deletions within the capsid (C) appears promising. C-deleted vaccines are able to replicate and secrete large amounts of non-infectious immunogenic subviral particles (SVPs) from transfected cells. We have previously generated a WNV DNA vaccine candidate pKUNdC/C where C-deleted WNV cDNA was placed under the control of one copy of the cytomegalovirus (CMV) promoter and the C gene was placed under the control of a second copy of the CMV promoter in the same plasmid DNA. This DNA was shown to generate single-round infectious particles (SRIPs) capable of delivering self-replicating C-deleted RNA producing SVPs to surrounding cells, thus enhancing the vaccine potential. However, the amounts of both SRIPs and SVPs produced from pKUNdC/C DNA were relatively low. In this investigation, we aimed at increasing SRIP production by optimizing trans-C expression via incorporating different forms of C and the use of a more powerful promoter. The construct containing an elongation factor EF1α promoter encoding an extended form of C was demonstrated to produce the highest titres of SRIPs and was immunogenic in mice. Additionally, SRIP and SVP titres were further improved via incorporation of a glycosylation motif in the envelope protein. The optimized DNA yielded ~100-fold greater titres of SRIPs than the original construct, thus providing a promising candidate for further vaccine evaluation.
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http://dx.doi.org/10.1099/vir.0.064121-0DOI Listing
October 2014

Loop de loop: viral RNA evades IFIT1 targeting.

Trends Microbiol 2014 Apr 13;22(4):171-3. Epub 2014 Mar 13.

Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, St Luca, Brisbane, Queensland 4072, Australia. Electronic address:

In a landmark finding published in Science, Hyde et al. have demonstrated that a hairpin RNA structure adjacent to the 5' cap of alphavirus genomic RNA confers the ability of these viruses to evade restriction by the interferon-induced host protein IFIT1.
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http://dx.doi.org/10.1016/j.tim.2014.02.014DOI Listing
April 2014

Noncoding subgenomic flavivirus RNA: multiple functions in West Nile virus pathogenesis and modulation of host responses.

Viruses 2014 Jan 27;6(2):404-27. Epub 2014 Jan 27.

Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane 4072, Australia.

Flaviviruses are a large group of positive strand RNA viruses transmitted by arthropods that include many human pathogens such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus, dengue virus, and tick-borne encephalitis virus. All members in this genus tested so far are shown to produce a unique subgenomic flavivirus RNA (sfRNA) derived from the 3' untranslated region (UTR). sfRNA is a product of incomplete degradation of genomic RNA by the cell 5'-3' exoribonuclease XRN1 which stalls at highly ordered secondary RNA structures at the beginning of the 3'UTR. Generation of sfRNA results in inhibition of XRN1 activity leading to an increase in stability of many cellular mRNAs. Mutant WNV deficient in sfRNA generation was highly attenuated displaying a marked decrease in cytopathicity in cells and pathogenicity in mice. sfRNA has also been shown to inhibit the antiviral activity of IFN-α/β by yet unknown mechanism and of the RNAi pathway by likely serving as a decoy substrate for Dicer. Thus, sfRNA is involved in modulating multiple cellular pathways to facilitate viral pathogenicity; however the overlying mechanism linking all these multiple functions of sfRNA remains to be elucidated.
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http://dx.doi.org/10.3390/v6020404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939463PMC
January 2014

West Nile virus genome with glycosylated envelope protein and deletion of alpha helices 1, 2, and 4 in the capsid protein is noninfectious and efficiently secretes subviral particles.

J Virol 2013 Dec 18;87(23):13063-9. Epub 2013 Sep 18.

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Australia.

Flavivirus genomes with deletions in the capsid (C) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (SVPs) without generating infectious virus. Here, we report that cytomegalovirus promoter-driven cDNA of West Nile virus Kunjin (KUNV) containing a glycosylation motif in the envelope (E) gene and a combined deletion of alpha helices 1, 2, and 4 in C produces significantly more SVPs than KUNV cDNAs with nonglycosylated E and various other deletions in C.
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http://dx.doi.org/10.1128/JVI.01552-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838115PMC
December 2013
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