We report for the first time the presence of phosphorylated transactivation response DNA‐binding protein of 43 kDa (p‐TDP‐43)‐immunoreactive cytoplasmic inclusions in Schwann cells in an autopsy case ...of sporadic amyotrophic lateral sclerosis (ALS). An 81‐year‐old woman with no family history of neuromuscular disorders noticed difficulty in handling chopsticks due to weakness of the hands. She then developed weakness of the lower and upper limbs and dyspnea. Neurological examination at the age of 83 years revealed disorientation, severe weakness of the facial muscles, tongue, neck and extremities, and fasciculations in the thighs. She exhibited hyperactive jaw jerk and lower limb deep tendon reflexes and normal upper limb deep tendon reflexes, and left extensor plantar response was observed. The patient was diagnosed as having sporadic ALS. An autopsy performed at the age of 84 years revealed widespread p‐TDP‐43‐immunoreactive neuronal and glial cytoplasmic inclusions in the cerebrum, brain stem, and spinal cord, in addition to some Bunina bodies. Moreover, a small number of p‐TDP‐43‐immunoreactive inclusions were found in the facial or accoustic nerve (indistinguishable), spinal cord anterior roots, cauda equina, and peripheral nerves in the dorsal root ganglia. Immunohistochemical staining for p‐TDP‐43 revealed just a few p‐TDP‐43‐immunoreactive inclusions surrounding axons in the cervical and lumbar anterior roots. Double immunofluorescence analysis revealed that these inclusions were co‐localized with S‐100 protein β, suggesting that these inclusions were localized in the cytoplasm of Schwann cells. The peripheral nervous system including Schwann cells may be involved in TDP‐43 pathology in ALS.
Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the ...form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.
The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.
The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments (vRNAs) are selectively packaged into progeny virions, with eight vRNAs in ...ribonucleoprotein complexes (RNPs) arranged in a specific "1+7" pattern, that is, one central RNP surrounded by seven RNPs. Here we report the genome packaging of an artificially generated seven-segment virus that lacks the hemagglutinin (HA) vRNA. Electron microscopy shows that, even in the presence of only seven vRNAs, the virions efficiently package eight RNPs arranged in the same "1+7" pattern as wild-type virions. Next-generation sequencing reveals that the virions specifically incorporate host-derived 18S and 28S ribosomal RNAs (rRNAs) seemingly as the eighth RNP in place of the HA vRNA. These findings highlight the importance of the assembly of eight RNPs into a specific "1+7" configuration for genome packaging in progeny virions and suggest a potential role for cellular RNAs in viral genome packaging.
The influenza A virus genome is composed of eight single-stranded negative-sense viral RNA segments (vRNAs). The eight vRNAs are selectively packaged into each progeny virion. This process likely ...involves specific interactions between the vRNAs via segment-specific packaging signals located in both the 3'- and 5'-terminal regions of the respective vRNAs. To assess the importance of vRNA-vRNA interactions via packaging signals for selective genome packaging, we generated mutant viruses possessing silent mutations in the packaging signal region of the hemagglutinin (HA) vRNA. A mutant virus possessing silent mutations in nucleotides (nt) 1664 to 1676 resulted in defects in HA vRNA incorporation and showed a reduction in viral growth. After serial passage, the mutant virus acquired additional mutations in the 5'-terminal packaging signal regions of both the HA and polymerase basic 2 (PB2) vRNAs. These mutations contributed to the recovery of viral growth and HA vRNA packaging efficiency. In addition, an RNA-RNA interaction between the 5' ends of HA and PB2 vRNAs was confirmed
, and this interaction was disrupted following the introduction of silent mutations in the HA vRNA. Thus, our results demonstrated that RNA-RNA interactions between the packaging signal regions of HA vRNA and PB2 vRNA are important for selective genome packaging.
While numerous viral genomes comprise a single genome segment, the influenza A virus possesses eight segmented genomes. Influenza A virus can benefit from having a segmented genome because the segments can reassort with other strains of the influenza virus to create new genetically distinct strains. The influenza A virus efficiently incorporates one copy of each of its eight genomic segments per viral particle. However, the mechanism by which each segment is specifically selected is poorly understood. The genome segments contain RNA signals that facilitate the incorporation of segments into virus particles. These regions may facilitate specific interactions between the genome segments, creating an eight-segment complex, which can then be packaged into individual particles. In this study, we provide evidence that RNA signals contribute to specific interactions between two of the influenza virus genome segments.
Herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) plays an essential role in viral entry. The functional regions of gD responsible for viral entry have been mapped to its extracellular ...domain, whereas the gD cytoplasmic domain plays no obvious role in viral entry. Thus far, the role(s) of the gD cytoplasmic domain in HSV-1 replication has remained to be elucidated. In this study, we show that ectopic expression of gD induces microvillus-like tubular structures at the plasma membrane which resemble the reported projection structures of the plasma membrane induced in HSV-1-infected cells. Mutations in the arginine cluster (residues 365 to 367) in the gD cytoplasmic domain greatly reduced gD-induced plasma membrane remodeling. In agreement with this, the mutations in the arginine cluster in the gD cytoplasmic domain reduced the number of microvillus-like tubular structures at the plasma membrane in HSV-1-infected cells. In addition, the mutations produced an accumulation of unenveloped nucleocapsids in the cytoplasm and reduced viral replication and cell-cell spread. These results suggest that the arginine cluster in the gD cytoplasmic domain is required for the efficient induction of plasma membrane projections and viral final envelopment, and these functions of the gD domain may lead to efficient viral replication and cell-cell spread.
The cytoplasmic domain of HSV-1 gD, an envelope glycoprotein essential for viral entry, was reported to promote viral replication and cell-cell spread, but the role(s) of the domain during HSV-1 infection has remained unknown. In this study, we clarify two functions of the arginine cluster in the HSV-1 gD cytoplasmic domain, both of which require host cell membrane remodeling, i.e., the formation of microvillus-like projections at the plasma membrane and viral final envelopment in HSV-1-infected cells. We also show that the gD arginine cluster is required for efficient HSV-1 replication and cell-cell spread. This is the first report clarifying not only the functions of the gD cytoplasmic domain but also identifying the gD arginine cluster to be the HSV-1 factor responsible for the induction of plasma membrane projections in HSV-1-infected cells. Our results elucidate some of the functions of this multifunctional envelope glycoprotein during HSV-1 infection.
Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral ...therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.
In order to investigate the novel function(s) of the herpes simplex virus 1 (HSV-1) immediate early protein ICP22, we screened for ICP22-binding proteins in HSV-1-infected cells. Our results were as ...follows. (i) Tandem affinity purification of ICP22 from infected cells, coupled with mass spectrometry-based proteomics and subsequent analyses, demonstrates that ICP22 forms a complex(es) with the HSV-1 proteins UL31, UL34, UL47 (or VP13/14), and/or Us3. All these proteins were previously reported to be important for viral egress through the nuclear membrane. (ii) ICP22 colocalizes with UL31 and UL34 at the nuclear membrane in wild-type HSV-1-infected cells. (iii) The UL31-null mutation prevents the targeting of ICP22 to the nuclear membrane. (iv) The ICP22-null mutation resulted in UL31 and UL34 being mislocalized in the endoplasmic reticulum (in addition to the nuclear membrane) and significantly reduced numbers of primary enveloped virions in the perinuclear space, although capsids accumulated in the nuclei. Collectively, these results suggest that (i) ICP22 interacts with HSV-1 regulators of nuclear egress, including UL31, UL34, UL47, and Us3 in HSV-1-infected cells; (ii) UL31 mediates the recruitment and anchorage of ICP22 at the nuclear membrane; and (iii) ICP22 plays a regulatory role in HSV-1 primary envelopment, probably by interacting with and regulating UL31 and UL34. Here we report a previously unknown function for ICP22 in the regulation of HSV-1 nuclear egress.
The herpes simplex virus 1 (HSV-1) immediate early protein ICP22 is recognized primarily as a regulator of viral gene expression. In this study, we show that ICP22 interacts with the HSV-1 proteins UL31 and UL34, which play crucial roles at the nuclear membrane in HSV-1 primary envelopment during viral nuclear egress. We also demonstrate that UL31 is required for the targeting of ICP22 to the nuclear membrane and that ICP22 is required for the correct localization of UL31 and/or UL34. Furthermore, we confirm that ICP22 is required for efficient HSV-1 primary envelopment during viral nuclear egress. Thus, we report, for the first time, that ICP22 plays a regulatory role in HSV-1 nuclear egress.
We herein report a 44-year-old Japanese man with hereditary transthyretin amyloidosis (ATTRv amyloidosis) harboring the variant Leu58Arg (p.Leu78Arg) in TTR in whom we conducted an observational ...study with liver transplantation (LT) and transthyretin (TTR) stabilizers (tafamidis and diflunisal) for 9 years. This patient showed gradual deterioration of sensory, motor, and autonomic neuropathy symptoms after LT. Furthermore, cardiac amyloidosis gradually developed. Although the present case showed deterioration of the symptoms after disease-modifying treatments, LT might be suitable in patients with the same variant if they are young and in good condition due to a long survival after LT.
Hepatitis C virus (HCV), dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the family Flaviviridae. Their viral particles have the envelope composed of viral proteins and a lipid ...bilayer acquired from budding through the endoplasmic reticulum (ER). The phospholipid content of the ER membrane differs from that of the plasma membrane (PM). The phospholipase A2 (PLA2) superfamily consists of a large number of members that specifically catalyse the hydrolysis of phospholipids at a particular position. Here we show that the CM-II isoform of secreted PLA2 obtained from Naja mossambica mossambica snake venom (CM-II-sPLA2) possesses potent virucidal (neutralising) activity against HCV, DENV and JEV, with 50% inhibitory concentrations (IC50) of 0.036, 0.31 and 1.34 ng/ml, respectively. In contrast, the IC50 values of CM-II-sPLA2 against viruses that bud through the PM (Sindbis virus, influenza virus and Sendai virus) or trans-Golgi network (TGN) (herpes simplex virus) were >10,000 ng/ml. Moreover, the 50% cytotoxic (CC50) and haemolytic (HC50) concentrations of CM-II-sPLA2 were >10,000 ng/ml, implying that CM-II-sPLA2 did not significantly damage the PM. These results suggest that CM-II-sPLA2 and its derivatives are good candidates for the development of broad-spectrum antiviral drugs that target viral envelope lipid bilayers derived from the ER membrane.
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