Michael Oglesbee

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded.

Measles virus is a negative‐sense, single‐stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N‐terminal domain, and of an intrinsically disordered C‐terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C‐terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in NTAIL, an α‐helical molecular recognition element (α‐MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small‐angle X‐ray scattering, we have derived a low‐resolution structural model of the complex between XD and NTAIL, which shows that most of NTAIL remains disordered in the complex despite P‐induced folding within the α‐MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N‐terminal region of NTAIL, and of a bulky globular region, corresponding to XD and to the C terminus of NTAIL (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that NTAIL has an additional site (aa 517–525) involved in binding to XD but not in the unstructured‐to‐structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.

Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein

ABSTRACT The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

Origin, Evolution, and Virulence of Porcine Deltacoronaviruses in the United States

ABSTRACT A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine. IMPORTANCE Porcine coronaviruses (CoVs) are major viral infectious diseases of swine. Examples of porcine CoVs include porcine transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine respiratory coronavirus (PRCV). In February 2014, another porcine CoV, porcine deltacoronavirus (PdCV), emerged in Ohio and Indiana and subsequently spread rapidly across the United States and Canada, causing significant economic losses. Here, we report the detailed genetic characterization, phylogeny, and virulence of emergent PdCV strains in the United States. We found that PdCV caused severe diarrhea, vomiting, and dehydration in gnotobiotic and conventional piglets, signs that were clinically indistinguishable from those caused by PEDV and TGEV. In addition to extensive intestinal lesions, PdCV caused significant lesions in the stomach and mild pulmonary lesions that have not been reported for TGEV and PEDV. The finding that PdCV is a significant enteric disease of swine highlights the need to develop effective measures to control this disease. Porcine coronaviruses (CoVs) are major viral infectious diseases of swine. Examples of porcine CoVs include porcine transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine respiratory coronavirus (PRCV). In February 2014, another porcine CoV, porcine deltacoronavirus (PdCV), emerged in Ohio and Indiana and subsequently spread rapidly across the United States and Canada, causing significant economic losses. Here, we report the detailed genetic characterization, phylogeny, and virulence of emergent PdCV strains in the United States. We found that PdCV caused severe diarrhea, vomiting, and dehydration in gnotobiotic and conventional piglets, signs that were clinically indistinguishable from those caused by PEDV and TGEV. In addition to extensive intestinal lesions, PdCV caused significant lesions in the stomach and mild pulmonary lesions that have not been reported for TGEV and PEDV. The finding that PdCV is a significant enteric disease of swine highlights the need to develop effective measures to control this disease.

The highly inducible member of the 70 kDa family of heat shock proteins increases canine distemper virus polymerase activity.

The cellular stress response is characterized by the production of heat shock proteins (HSP) which serve important cytoprotective functions. Paradoxically, in vitro induction of the stress response promotes cytopathic effect mediated by infection with canine distemper virus (CDV). The stress-mediated increase in cytopathic effect is correlated to the formation of complexes between the viral nucleocapsid (NC) and the major inducible member of the approximately 70 kDa family of HSP (hsp72). The objective of the present study was to document the functional significance of CDV NC-HSP interaction. Cytoplasmic NC was purified from Vero cells lytically infected with the Onderstepoort strain of CDV. Both ultrastructural variants of CDV NC interacted with both hsp72 and the constitutively expressed member of the approximately 70 kDa family of HSP (hsp73) in a reversible and ATP-dependent manner. An effect of hsp72/73 on NC polymerase activity was demonstrated using cell-free assays derived from either Vero or HeLa cell lines. Antibody specific to hsp72 suppressed both basal and stress-enhanced polymerase activity whereas hsp73-specific antibody had no affect. Supplementation of purified hsp72/73, but not hsp73 alone, enhanced basal polymerase activity in a dosage-dependent manner. Using purified NC variants, polymerase activity was demonstrated in pre-formed hsp72/73-NC complexes but not in NC devoid of HSP. These results suggest that the stimulatory effect of the stress response upon CDV gene expression may, in part, be mediated by a reversible and direct interaction between hsp72 and the viral core particle.

Constitutive overexpression of the major inducible 70 kDa heat shock protein mediates large plaque formation by measles virus.

Induction of the cellular stress response elevates cytoplasmic levels of heat shock proteins (HSPs) belonging to multiple families. When infected with canine distemper virus or measles virus (MV), cells containing elevated HSPs support increased viral gene expression and cytopathic effect. The present work tests the hypothesis that increases in the major inducible 70 kDa HSP (hsp72) are sufficient to mediate the effect of stress response induction on infection phenotype. Human astrocytoma cells (U373) were stably transfected with the human hsp72 gene under control of the beta-actin promoter. Constitutive overexpression of hsp72 was demonstrated in multiple clones by Western blot analysis of cytoplasmic total protein. Southern blot analysis of cell DNA confirmed the recovery of genetically distinct clones. Infection of these clonal populations with MV resulted in increased viral transcript production relative to infected control cell lines. Increased transcript production was associated with increased viral membrane glycoprotein expression and cytopathic effect (i.e., mean plaque area). Increases in cytopathic effect were due to the emergence of a large plaque phenotype from a small plaque-purified inoculum, mimicking the effect of cellular stress response induction upon viral infection phenotype. Large plaque phenotypic variants reported in the literature are associated with enhanced neurovirulence, a fact that highlights the potential significance of physiologic elevations in hsp72 (e.g., fever-induced) that accompany in vivo viral infection.

Clinical Characterization of a Familial Degenerative Myelopathy in Pembroke Welsh Corgi Dogs

BACKGROUND Adult dogs with degenerative myelopathy (DM) have progressive ataxia and paresis of the pelvic limbs, leading to paraplegia and euthanasia. Although most commonly reported in German Shepherd dogs, high disease prevalence exists in other breeds. OBJECTIVE Our aim was the clinical and histopathologic characterization of familial degenerative myelopathy (FDM) in Pembroke Welsh Corgi (PWC) dogs. ANIMALS Twenty-one PWCs were prospectively studied from initial diagnosis until euthanasia. METHODS Neurologic examination, blood tests, cerebrospinal fluid (CSF) analysis, electrodiagnostic testing, and spinal imaging were performed. Concentrations of 8-iso-prostaglandin F2alpha (8-isoprostane) were measured in CSF. Routine histochemistry was used for neuropathology. Deoxyribonucleic acid and pedigrees were collected from 110 dogs. RESULTS Median duration of clinical signs before euthanasia was 19 months. Median age at euthanasia was 13 years. All dogs were nonambulatory paraparetic or paraplegic, and 15 dogs had thoracic limb weakness at euthanasia. Electrodiagnostic testing and spinal imaging were consistent with noncompressive myelopathy. No significant difference was detected in 8-isoprostane concentrations between normal and FDM-affected dogs. Axonal and myelin degeneration of the spinal cord was most severe in the dorsal portion of the lateral funiculus. Pedigree analysis suggested a familial disease. CONCLUSIONS AND CLINICAL IMPORTANCE Clinical progression of FDM in PWC dogs was similar to that observed in other breeds but characterized by a longer duration. Spinal cord pathology predominates as noninflammatory axonal degeneration. Oxidative stress injury associated with 8-isoprostane production is not involved in the pathogenesis of FDM-affected PWC dogs. A familial disease is suspected.

Role for Heat Shock Proteins in the Immune Response to Measles Virus Infection

Heat shock proteins (HSPs) are recognized for their support of protein metabolism. Interaction with viral proteins also enhances the development of innate and adaptive immune responses against the infecting agent. At the level of the infected cell, HSPs are uniquely expressed on the cell surface, where they represent targets of lymphokine activated killer cells. Necrosis of the infected cell releases complexes of HSP and viral protein, which, in turn, binds antigen-presenting cells (APCs). One effect of binding is to stimulate APC maturation and the release of proinflammatory cytokines, an adjuvant effect that prepares the way for adaptive immune responses. A second effect of binding is to direct the antigenic cargo of the HSP into endogenous MHC presentation pathways for priming of naive cytotoxic T cells (CTL) or activation of antigen-specific CTLs. This alternate pathway of antigen presentation is essential to CTL priming following primary brain infection. Using heat shock to elevate brain levels of HSP in a mouse model of measles virus (MV) persistent infection, we provide evidence supporting a role for HSPs in promoting cell-mediated viral clearance from brain. The findings highlight the probable relevance of HSPs to anti-MV immunity, suggesting novel routes of both therapeutic intervention and preventative measures.

hsp72, a Host Determinant of Measles Virus Neurovirulence

ABSTRACT Transient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.

Use of surface plasmon resonance for the measurement of low affinity binding interactions between HSP72 and measles virus nucleocapsid protein

The 72 kDa heat shock protein (HSP72) is a molecular chaperone that binds native protein with low affinity. These interactions can alter function of the substrate, a property known as HSP-mediated activity control. In the present work, BIAcore instrumentation was used to monitor binding reactions between HSP72 and naturally occurring sequence variants of the measles virus (MV) nucleocapsid protein (N), a structural protein regulating transcription/replication of the viral genome. Binding reactions employed synthetic peptides mimicking a putative HSP72 binding motif of N. Sequences were identified that bound HSP72 with affinities comparable to well-characterized activity control reactions. These sequences, but not those binding with lesser affinity, supported HSP72 activity control of MV transcription/replication. BIAcore instrumentation thus provides an effective way to measure biologically relevant low affinity interactions with structural variants of viral proteins.

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (NTAIL) are both intrinsically disordered. Here we show that Henipavirus NTAIL domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (PXD) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that NTAIL and PXD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 m and has a KD in the μm range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that PXD triggers an increase in the α-helical content of NTAIL. Using fluorescence spectroscopy, we show that PXD has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus NTAIL domain, thus arguing for the lack of stable contacts between the C termini of NTAIL and PXD. Finally, we present a tentative structural model of the NTAIL-PXD interaction in which a short, order-prone region of NTAIL (α-MoRE; amino acids 473–493) adopts an α-helical conformation and is embedded between helices α2 and α3 of PXD, leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the NTAIL-PXD interaction as a valuable target for rational antiviral approaches.