Minoru Tobiume

Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer

Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by de novo sequencing using a next-generation DNA sequencer on total RNAs extracted from an autopsy lung of a patient who died of viral pneumonia with A/H1N1/2009. Among a total of 9.4×106 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than ×200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly172Glu and Gly239Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.

Cutting Edge: Brucella abortus Exploits a Cellular Prion Protein on Intestinal M Cells as an Invasive Receptor

Brucella abortus is a Gram-negative bacterium causing brucellosis. Although B. abortus is known to infect via the oral route, the entry site in the gastrointestinal tract has been unclear. We found that B. abortus was selectively internalized by microfold cells (M cells), a subset of epithelial cells specialized for mucosal Ag uptake. During this process, colocalization of cellular prion protein (PrPC) and B. abortus was evident on the apical surface as well as in subapical vacuolar structures in M cells. Internalization of B. abortus by M cells of PrPC-deficient (Prnp−/−) mice was greatly reduced compared with that in wild-type mice. Furthermore, an oral infection study revealed that translocation of B. abortus into the Peyer’s patch was significantly lower in Prnp−/− than in wild-type mice. These observations suggest that orally infected B. abortus invades the host through M cells by using PrPC on the apical surface of M cells as an uptake receptor.

Establishment of a standard 14-3-3 protein assay of cerebrospinal fluid as a diagnostic tool for Creutzfeldt–Jakob disease

Periodic sharp wave complexes observed on an electroencephalographic recording and the presence of a 14-3-3 protein in the cerebrospinal fluid (CSF) are both included in the diagnostic criteria for the Creutzfeldt–Jakob disease (CJD) supplied by the World Health Organization; however, the presence or absence of the 14-3-3 protein in the CSF is sometimes difficult to discern on a western blot because of equivocal bands. The goal of this study was to establish a standard 14-3-3 protein assay and to determine the threshold level of a 14-3-3 protein that can be assayed by western blot. We searched for the most suitable isoform of the 14-3-3 protein to test for in protein assays, and the most sensitive antibody among four antibodies with an affinity for 14-3-3. We measured the levels of all 14-3-3 isoforms in 112 patients with CJD and in 100 patients with other diseases. We compared the performances of four different antibodies. We carried out a semi-quantitative analysis of γ-isoform levels using the LAS 3000 system, which was capable of producing a digital image from the luminescence on a western blot. We determined that the most suitable isoform of the 14-3-3 protein for conducting a standardized assay was the γ-isoform. Among the four commercially available antibodies for this protein, the most sensitive and specific was 18647 (IBL, Japan). We report the high repeatability of the detection of the 14-3-3 protein by this antibody to the γ-isoform, showing that western blot can be used for semi-quantitative analysis.

MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the recently discovered MARCH family of RING (really interesting new gene)-finger E3 ubiquitin ligases. MARCH8 downregulates several host transmembrane proteins, including major histocompatibility complex (MHC)-II, CD86, interleukin (IL)-1 receptor accessory protein, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 and the transferrin receptor. However, its physiological roles remain largely unknown. Here we identify MARCH8 as a novel antiviral factor. The ectopic expression of MARCH8 in virus-producing cells does not affect levels of lentivirus production, but it does markedly reduce viral infectivity. MARCH8 blocks the incorporation of HIV-1 envelope glycoprotein into virus particles by downregulating it from the cell surface, probably through their interaction, resulting in a substantial reduction in the efficiency of viral entry. The inhibitory effect of MARCH8 on vesicular stomatitis virus G-glycoprotein is even more remarkable, suggesting a broad-spectrum inhibition of enveloped viruses by MARCH8. Notably, the endogenous expression of MARCH8 is high in monocyte-derived macrophages and dendritic cells, and MARCH8 knockdown or knockout in macrophages significantly increases the infectivity of virions produced by these cells. Our findings thus indicate that MARCH8 is highly expressed in terminally differentiated myeloid cells, and that it is a potent antiviral protein that targets viral envelope glycoproteins and reduces their incorporation into virions.

Rabies virus dissemination in neural tissues of autopsy cases due to rabies imported into Japan from the Philippines: immunohistochemistry.

Two Japanese men, 65 and 69 years old, developed rabies in Japan around 2–3 months after dog‐bite exposure in the Philippines. Laboratory diagnosis of rabies was made following the detection of rabies virus genome on reverse transcription–polymerase chain reaction from saliva, and on immunohistochemistry of a nuchal skin punch biopsy in one case. The patients died 9 and 19 days after clinical onset. At autopsy, no macroscopic changes in the CNS were observed. Histopathology indicated that eosinophilic and cytoplasmic inclusion bodies, Negri bodies, were seen in neuronal cells of the CNS. Inflammatory cell reactions were scarce, and no apoptosis in the CNS was detected. Immunohistochemistry demonstrated that rabies virus nucleoprotein (N) and phosphoprotein (P) were disseminated to all neural tissues and cells in the body with a similar pattern in both cases. Interestingly, there were no differences of localization between N and P antigen in the brain, but the N antigen was located at the peripheral nerve sheaths and the P antigen was localized in axons. These data indicate that rabies virus dissemination in all neural tissues causes disease development and death. Immunohistochemistry for rabies is a powerful tool to understand the pathogenesis of rabies.

Accumulation of cellular prion protein within dystrophic neurites of amyloid plaques in the Alzheimer's disease brain

Amyloid plaques, a well‐known hallmark of Alzheimers disease (AD), are formed by aggregated β‐amyloid (Aβ). The cellular prion protein (PrPc) accumulates concomitantly with Aβ in amyloid plaques. One type of amyloid plaque, classified as a neuritic plaque, is composed of an amyloid core and surrounding dystrophic neurites. PrPc immunoreactivity reminiscent of dystrophic neurites is observed in neuritic plaques. Proteinase K treatment prior to immunohistochemistry removes PrPc immunoreactivity from amyloid plaques, whereas Aβ immunoreactivity is enhanced by this treatment. In the present study, we used a chemical pretreatment by a sarkosyl solution (0.1% sarkosyl, 75 mM NaOH, 2% NaCl), instead of proteinase K treatment, to evaluate PrPc accumulation within amyloid plaques. Since PrPc within amyloid plaques is removed by this chemical pretreatment, we can recognize that the PrP species deposits within amyloid plaques were PrPc. We could observe that PrPc accumulation in dystrophic neurites occurred differently compared with Aβ or hyperphosphorylated tau aggregation in the AD brain. These results could support the hypothesis that PrPc accumulation in dystrophic neurites reflects a response to impairments in cellular degradation, endocytosis, or transport mechanisms associated with AD rather than a non‐specific cross‐reactivity between PrPc and aggregated Aβ or tau.

Ultrasensitive detection of PrP(Sc) in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrP(Sc)) in the central nervous system. The pathological features and biochemical properties of PrP(Sc) in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrP(Sc). This method involves amplifying PrP(Sc) by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrP(C) substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrP(Sc) in the CSF by serial PMCA, and the CSF levels of PrP(Sc) tended to increase with disease progression. In addition, PrP(Sc) was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrP(Sc) in non-human primate models of CJD.

Identification and structural analysis of C-terminally truncated collapsin response mediator protein-2 in a murine model of prion diseases.

BackgroundPrion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrPSc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood.ResultsIn a proteomic analysis of soluble proteins in the brains of mice challenged intracerebrally with scrapie prion (Obihiro I strain), we found that the amount of the full-length form of collapsin response mediator protein-2 (CRMP-2; 61 kDa) decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-ΔC) lacked the sequence from serine518 to the C-terminus, including the C-terminal phosphorylation sites important for the regulation of axonal growth and axon-dendrite specification in developing neurons. The invariable size of the mRNA transcript in Northern blot analysis suggested that the truncation was due to post-translational proteolysis. By overexpression of CRMP-2-ΔC in primary cultured neurons, we observed the augmentation of the development of neurite branch tips to the same levels as for CRMP-2T514A/T555A, a non-phosphorylated mimic of the full-length protein. This suggests that the increased level of CRMP-2-ΔC in the brain modulates the integrity of neurons, and may be involved in the pathogenesis of the neuronal abnormalities observed in the late stages of the disease.ConclusionsWe identified the presence of CRMP-2-ΔC in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-ΔC in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase

ABSTRACT Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.

Simian fetal brain progenitor cells for studying viral neuropathogenesis

The pathogenesis of neurologic dysfunctions caused by human immunodeficiency virus type 1 (HIV-1) infection is not yet well understood. Simian immunodeficiency virus (SIV) infection of macaques is an important animal model for HIV-1 infection. This is the first report to characterize brain progenitor cells (BPCs) isolated from embryonic brain of cynomolgus monkeys (Macaca fascicularis) by neurosphere assay and utilize BPC-derived cell culture for studying SIV infection. The self-renewal and multilineage differentiation properties of BPCs are convenient for planning viral infection experiments. The BPC-derived culture does not contain macrophage/microglial cells, fibroblasts, or endothelial cells. Thus, this culture is appropriate for studying direct relation between SIV infection and neuronal and glial cells. First, the authors characterized undifferentiated and differentiated simian BPCs by immunocytochemistry, flow cytometry analysis, real-time polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR. The BPCs induced to differentiate by the addition of 1% fetal bovine serum (FBS) were composed of heterogeneous cells expressing nestin, glial fibrillary acidic protein (GFAP), and/or tubulin beta III isoform (Tuj). None of them expressed the monocyte/macrophage/microglial marker. mRNA expression of CD4, CXCR4, CCR5, GPR1, STRL33, and APJ in both undifferentiated and differentiated BPCs were shown by RT-PCR method, suggesting that SIV would infect and replicate in this culture system. Then, it was confirmed that the neurotropic SIV strain, SIV17/E-Fr, replicated productively in BPC-derived cells. The SIV/17E-FrΔnefGFP was inoculated to identify the infected cells and immunocytochemistry analysis revealed that green fluorescent protein (GFP)-expressing cells were mostly GFAP positive and coexpressed with SIV p27 antigen. Thus, BPC-derived cell culture system is applicable for studying SIV infection in glial and neuronal cells.