Human parainfluenza virus type 2 V protein inhibits mitochondrial apoptosis pathway through two ways.

Paramyxoviruses are reported to block apoptosis for their replication, but the mechanisms remain unclear. Furthermore, regulation of mitochondrial apoptosis by paramyxoviruses has been hardly reported. We investigated whether and how human parainfluenza virus type 2 (hPIV-2) counteracts apoptosis. Infection of recombinant hPIV-2 carrying mutated V protein showed higher caspase 3/7 activity and higher cytochrome c release from mitochondria than wild type hPIV-2 infection. This indicates that V protein controls mitochondrial apoptosis pathway. hPIV-2 V protein interacted with Bad, an apoptotic promoting protein, and this interaction inhibited the binding of Bad to Bcl-XL. V protein also bound to 14-3-3ε, which was essential for inhibition of 14-3-3ε cleavage. Our data collectively suggest that hPIV-2 V protein has two means of preventing mitochondrial apoptosis pathway: the inhibition of Bad-Bcl-XL interaction and the suppression of 14-3-3ε cleavage. This is the first report of the mechanisms behind how paramyxoviruses modulate mitochondrial apoptosis pathways.

Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells. IMPORTANCE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.

The bipartite promoter of Orthonairovirus hazaraense large segment.

IMPORTANCE: The realization that segmented negative-strand RNA virus genome ribonucleoproteins are never free as their RNA ends are always bound to the viral polymerase has highlighted the problem of how these genome segments are replicated and express their mRNAs while their RNA ends remain associated with the polymerase throughout the cycles of RNA synthesis. This study of the length and nucleotide composition of the Orthonairovirus hazaraense L segment-specific double-stranded RNA (dsRNA) promoter element (the promoter duplex) provides insight into how its mRNA might be initiated and suggests that this promoter element acts via its separated single strands as well as via dsRNA.

Hazara orthonairovirus nucleoprotein facilitates viral cell-to-cell spread by modulating tight junction protein, claudin-1.

Background: Tight junctions act as a barrier that prevents invasion of pathogens through epithelial cells. This study aims to elucidate the correlation between tight junctions and nairoviruses using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. Methods: mRNA, total protein, and cell surface protein levels of tight junction proteins were examined by quantitative real-time reverse transcription polymerase chain reaction, immunoblot and flow cytometry, respectively. HAZV growth was measured by plaque assay. Immunofluorescence assay was used to examine viral cell-to-cell spread. The interaction between HAZV nucleoprotein and claudin-1 was analyzed by immunoprecipitation. Results: HAZV infection induced mRNA of several tight junction proteins, especially claudin-1. HAZV infection also induced cell surface expression of claudin-1 protein. Claudin-1 overexpression inhibited the growth of HAZV by blocking its cell-to-cell spread. In contrast, HAZV nucleoprotein completely inhibited HAZV-induced cell surface expression of claudin-1, and this inhibition required interaction between HAZV nucleoprotein and claudin-1. Conclusion: HAZV nucleoprotein was shown to bind to claudin-1 to negatively regulate its cell surface expression, and so can promote cell-to-cell spread of HAZV. This is the first presentation of a possible mechanism behind how nairoviruses counteract tight junction barrier function.

Hazara Orthonairovirus Nucleoprotein Antagonizes Type I Interferon Production by Inhibition of RIG-I Ubiquitination.

Viruses have evolved various strategies to evade the host innate immune system. The relationship between nairoviruses and the interferon (IFN) system is poorly understood. We investigated whether and how nairoviruses antagonize host innate immunity using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. HAZV nucleoprotein (N) was found to interact with the tripartite motif-containing protein 25 (TRIM25). The N-terminal region of N protein and the C-terminal region of TRIM25 are important for their interaction. Overexpression of N protein results in weakened interaction of TRIM25 with retinoic acid-inducible gene I (RIG-I). Furthermore, K63-linked polyubiquitination of RIG-I is inhibited in the presence of N protein. Our data collectively suggest that HAZV N protein interferes with the binding of TRIM25 to RIG-I and subsequent K63-linked polyubiquitination of RIG-I, which leads to inhibition of type I IFN production.

A Point Mutation in the Human Parainfluenza Virus Type 2 Nucleoprotein Leads to Two Separate Effects on Virus Replication.

Paramyxovirus genomes, like that of human parainfluenza virus type 2 (hPIV2), have lengths of precisely multiples-of-six nucleotides ("rule of six"), where each nucleoprotein subunit (NP) binds exactly six nucleotides. Ten residues of its RNA binding groove contact the genome RNA; but only one, Q202, directly contacts a nucleotide base. The mutation of NPQ202 leads to two phenotypes: the ability of the viral polymerase to replicate minigenomes with defective bipartite promoters where NPwt is inactive, and the inability to rescue rPIV2 carrying this point mutation by standard means. The absence of an rPIV2 NPQ202A prevented further study of the latter phenotype. By extensive and repeated cocultivation of transfected cells, an rPIV2 carrying this mutation was finally recovered, and this virus was apparently viable due to the presence of an additional NP mutation (I35L). Our results suggest that these two phenotypes are due to separate effects of the Q202 mutation, and that the problematic rescue phenotype may be due to the inability of the transfected cell to incorporate viral nucleocapsids during virus budding. IMPORTANCE Paramyxovirus genomes are contained within a noncovalent homopolymer of its nucleoprotein (NP) and form helical nucleocapsids (NC) whose 3' ends contain the promoters for the initiation of viral RNA synthesis. This work suggests that these NC 3' ends may play another role in the virus life cycle via their specific interaction with virus-modified cell membranes needed for the incorporation of viral NCs into budding virions.

Human Parainfluenza Virus Type 2 V Protein Modulates Iron Homeostasis.

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.IMPORTANCE hPIV-2 V protein interferes with interaction between FTH1 and NCOA4 and inhibits NCOA4-mediated ferritin degradation, leading to the inhibition of iron release to the cytoplasm. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.

Relationship between the induced-fit loop and the activity of Klebsiella pneumoniae pullulanase.

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706-710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706-710 and alters the binding affinity of the substrate.

Transfer of the Lower Trapezius as a Surgical Treatment for Combined Injuries to the Suprascapular and Axillary Nerves: A Case Report.

INTRODUCTION: Combined injuries to the suprascapular and axillary nerves can result in irreversible dysfunction of the shoulder joint, with reconstruction of shoulder external rotation being an essential component of an effective treatment. Transfer of the lower portion of the trapezius to the infraspinatus has been used, with success, to regain external rotation of the shoulder. CASE REPORT: We present the case of a 45-year-old man with a chronic traumatic injury of the suprascapular and axillary nerves. In addition to a surgical transfer of the lower trapezius to the infraspinatus, we included a transfer of the latissimus dorsi and teres major, with a tensor fasciaelatae graft to the supraspinatus tendon insertion, to improve the muscular strength of shoulder elevation and abduction, as well as to improve external rotation. At 24-month post-surgery, the patient had recovered 170° of shoulder elevation, 170° of abduction, and 60° of external rotation. CONCLUSION: Early recovery after surgery was achieved, with excellent improvement of the range of shoulder motion. We report the transfer of the lower trapezius to the infraspinatus might provide a useful salvage procedure for patients with poor functional prognosis of a chronic suprascapular nerve injury.

Increase in the thermostability of GH11 xylanase XynJ from Bacillus sp. strain 41M-1 using site saturation mutagenesis.

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a ß-jellyroll fold composed of eight ß strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the ß strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the ß-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.

High-resolution crystal structures of the glycoside hydrolase family 45 endoglucanase EG27II from the snail Ampullaria crossean.

Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Šresolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double-ψ six-stranded ß-barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high-resolution structures of EG27II provide a large amount of information for structure-based enzyme modification and cell-surface engineering, which will advance biofuel production using animal-derived cellulases.

Identification of key residues for activities of atypical glutathione S-transferase of Ceriporiopsis subvermispora, a selective degrader of lignin in woody biomass, by crystallography and functional mutagenesis.

Ceriporiopsis subvermispora (C. subvermispora) is a selective degrader of lignin in the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in cellular detoxification and metabolism. The crystal structures of a GST of C. subvermispora, CsGST83044, in GSH-free and -bound forms were solved at 1.95 and 2.19 Šresolution, respectively. The structure of the GSH-bound form revealed that CsGST83044 can be categorized as an atypical-type of GST. In the GSH-bound form of CsGST83044, Asn22, Asn24, and Tyr46 are located closest to the sulfur atom and form hydrogen bonds with the thiol group. The functional mutagenesis indicated that they are critical for the enzymatic activities of CsGST83044. The critical residues of an atypical-type GST belonging to the GSTFuA class were revealed for the first time. A previous study indicated that CsGST83044 and another GST, CsGST63524, differ in substrate preference; CsGST83044 prefers smaller substrates than CsGST63524 for its esterase activity. The GSH-bound pocket of CsGST83044 turns out to be small, which may explain the preference for smaller substrates. Protein engineering of GSTs of C. subvermispora in the light of the obtained insight may pave a path in the future for utilization of the woody biomass.

Structure of a serine-type glutathione S-transferase of Ceriporiopsis subvermispora and identification of the enzymatically important non-canonical residues by functional mutagenesis.

Ceriporiopsis subvermispora (C. subvermispora), one of the white-rot fungi, is known as a selective lignin degrader of the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that are capable of catalyzing the reactions involved in detoxification and metabolic pathways. In this study, a GST of C. subvermispora, named CsGST63524, was overexpressed in E. coli, and then purified by affinity, anion exchange, and size exclusion column chromatography. The crystal structures of the CsGST63524 in ligand-free and complex with GSH were refined at 2.45 and 2.50 Šresolutions, respectively. The sulfur atom of glutathione forms a hydrogen bond with Ser21 of CsGST63524, indicating it is a serine-type GST. Mutagenesis of Ser21 unexpectedly indicated that this serine residue is not essential for the enzymatic activity of CsGST63524. Comparative sequence and structural analyses, together with functional mutagenesis, newly identified the enzymatically important non-canonical amino acid residues, Asn23 and Tyr45, other than the serine residue.

Elucidation of the mechanism of interaction between Klebsiella pneumoniae pullulanase and cyclodextrin.

Crystal structures of Klebsiella pneumoniae pullulanase (KPP) in complex with α-cyclodextrin (α-CD), ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) were refined at around 1.98-2.59 Šresolution from data collected at SPring-8. In the structures of the complexes obtained with 1 mM α-CD or γ-CD, one molecule of CD was found at carbohydrate-binding module 41 only (CBM41). In the structures of the complexes obtained with 1 mM ß-CD or with 10 mM α-CD or γ-CD, two molecules of CD were found at CBM41 and in the active-site cleft, where the hydrophobic residue of Phe746 occupies the inside cavity of the CD rings. In contrast to α-CD and γ-CD, one ß-CD molecule was found at the active site only in the presence of 0.1 mM ß-CD. These results were coincident with the solution experiments, which showed that ß-CD inhibits this enzyme more than a thousand times more potently than α-CD and γ-CD. The strong inhibition of ß-CD is caused by the optimized interaction between ß-CD and the side chain of Phe746. The increased Ki values of the F746A mutant for ß-CD supported the importance of Phe746 in the strong interaction of pullulanase with ß-CD.

Is a pulling sensation in the anteroposterior direction associated with otolith dysfunction?

CONCLUSION: A pulling sensation in the anteroposterior direction is suggested to originate from a dysfunction of the otolith organs. OBJECTIVES: Previous study with vestibular evoked myogenic potential (VEMP) confirmed that a falling sensation (in an up or down direction) and a lateral tilt sensation (in a right or left direction) were caused by otolith lesions. The purpose of this study was to clarify whether a pulling sensation in the anteroposterior (forward or backward) direction originates from otolith dysfunction. METHODS: The otolith function was assessed by cervical and ocular VEMPs (cVEMPs and oVEMPs) in 12 patients who complained of a forward or backward pulling sensation. cVEMPs were evaluated by the asymmetry ratio (AR) of the amplitude of the p13-n23 wave and the peak latencies of the p13 and n23 waves. oVEMPs were evaluated by the AR of the amplitude of the n1-p1 wave and the peak latency of the n1 and p1 waves. RESULTS: Abnormal ARs on cVEMP were observed in 7 of 12 patients. Nine of 12 patients had abnormal oVEMP results including 3 bilateral absent responses. Most (10 of 12) patients had abnormal cVEMP and/or oVEMP results. The latency of each detected wave was within the normal ranges.

Detection of utricular dysfunction using ocular vestibular evoked myogenic potential in patients with benign paroxysmal positional vertigo.

BACKGROUND: The ocular vestibular evoked myogenic potential (oVEMP) is thought to originate from the contralateral utricular organ. However, the clinical use of oVEMP has not yet been established. This study aimed to clarify whether oVEMP could be used to detect utricular dysfunction in patients with benign paroxysmal positional vertigo (BPPV). MATERIALS AND METHODS: Sixteen patients with BPPV underwent oVEMP measurements. Recordings were made on 2 separate occasions: when typical nystagmus was confirmed (pretreatment oVEMP) and 1 week after performing Epley's maneuver (posttreatment oVEMP). Results were evaluated using the asymmetry ratio (AR) of n1-p1 wave peak-to-peak amplitude and defined as reduced oVEMP when AR was >31.6%, or augmented oVEMP when AR was <-31.6%. RESULTS: Bilateral responses were recorded in 13 patients on the pretreatment oVEMP. Abnormal results were found in 11 patients (84.5%). These included 5 patients with reduced response and 6 with augmented response. On the posttreatment oVEMP, abnormal results were found in 5 patients (38.5%). All indicated reduced oVEMP. Abnormal results on the pretreatment oVEMP were not related to any persistent positional vertigo (p>0.05, Fisher's exact test). Three out of 4 patients (75.0%) with continuing unsteadiness had abnormal results (reduced response) on the posttreatment oVEMP. DISCUSSION: The oVEMP measurements indicated abnormal function of the utricle in patients with BPPV. Reduced oVEMP is thought to originate from the partial degeneration of utricular hair cells. Conversely, augmented oVEMP in the affected ear is thought to originate from a hypermobility of the stereocilia due to the detachment of otoconia within the utricle. The above-mentioned utricular dysfunction should be independent of the existence of otoconia in the semicircular canal; thus, the results of oVEMP were not related to the recovery of symptoms. CONCLUSION: oVEMP can be reliably used to detect utricular lesions in patients with BPPV.

Analysis of benign paroxysmal positional nystagmus in children.

INTRODUCTION: Benign paroxysmal positional vertigo (BPPV) is the most common vestibular disorder. However, BPPV in children has been studied less extensively than in the adult population. This is because the observation of benign paroxysmal positional nystagmus (BPPN) in children is technically very difficult and BPPV is rare in children. In this study, we present the only two cases of BPPV in children in which we successfully recorded and analyzed the BPPN. METHODS: One case was an 11-year-old boy and the other was a 3-year-old girl. We analyzed their BPPN three-dimensionally. RESULTS: Apogeotropic positional nystagmus was observed in the first case. We analyzed it to verify the presence of cupulolithiasis in the horizontal semicircular canal (HSCC). Geotropic positional nystagmus was observed in the second case, and the analyzed data indicated the presence of canalolithiasis in HSCC. Over the last decade, we have examined 3341 patients complaining of vertigo or dizziness. Among them, there were 63 children with the same complaint, so that the proportion of cases of BPPV in children was only 3% (2/63). DISCUSSION: Among patients complaining of vertigo or dizziness, children with BPPV are rare (3%). However, we have recorded their BPPN to confirm that BPPV does occur in children and that their characteristics of positional nystagmus are generally identical to those in adults. We emphasize that this is the first report of a child as young as 3 years old being diagnosed with BPPV.

Vestibular-evoked myogenic potential in response to bone-conducted sound in patients with otosclerosis.

CONCLUSION: Saccular dysfunction is a major cause of balance problems in patients with otosclerosis. Vestibular-evoked myogenic potential in response to bone-conducted sound (BC-VEMP) testing is useful for diagnosis of these patients. OBJECTIVES: The purpose of this study was to elucidate the origin of balance problems in patients with otosclerosis using BC-VEMP. METHODS: Subjects comprised 25 patients with unoperated otosclerosis (9 men and 16 women). They were divided into two groups depending on type of balance problems. Results of cochleo-vestibular functions including pure-tone audiometry, caloric testing, and BC-VEMP testing were compared between the two groups. RESULTS: Ten patients had complained of dizziness and/or vertigo (disequilibrium group), and the other 15 patients had not (Non-disequilibrium group). Nine patients showed abnormal results on BC-VEMP testing in the disequilibrium group, while one patient had abnormal results in the non-disequilibrium group (p < 0.001).

Furosemide-loading vestibular evoked myogenic potential testing can suggest developing bilateral involvement of unilateral Meniere's disease.

CONCLUSIONS: Patients with positive results of furosemide-loading vestibular evoked myogenic potential (F-VEMP) testing in the unaffected ears of unilateral Meniere's disease have a high incidence of developing bilateral lesions. OBJECTIVE: To clarify the meaning of positive results of F-VEMP testing of the unaffected ear of patients with unilateral Meniere's disease. METHODS: Twenty-five patients with unilateral Meniere's disease were investigated in this study. The positive group consisted of 6 patients with positive results of F-VEMP testing in the contralateral ear and the negative group consisted of 19 patients with negative results. The incidence of contralateral involvement was compared in both groups by the Kaplan-Meier method. RESULTS: Contralateral involvement was seen in three cases (50%) in the positive group after 2, 12, and 26 months and in three cases (16%) in the negative group after 27, 56, and 78 months. The positive group had a higher incidence of contralateral involvement than the negative group (p = 0.0017, according to a log-rank test).