Masae Itoh

Longer and Shorter Forms of Sendai Virus C Proteins Play Different Roles in Modulating the Cellular Antiviral Response

ABSTRACT The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-α from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-α signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.

The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon

The non-structural protein 5A (NS5A) of some hepatitis C virus (HCV) isolates has been implicated in the inhibition of the antiviral activity of interferon (IFN). In the present study, the possible inhibitory effects of NS5A from two isolates of HCV subtype 1b, HCV-1bJk and M094AJk, and their chimeric form on the antiviral activity of IFN were examined. HCV-1bJk and M094AJk are categorized as IFN resistant and IFN sensitive, respectively, based on the sequences of the IFN-sensitivity determining region (ISDR). When encephalomyocarditis virus was used as a challenge virus, NS5A was shown to eliminate the antiviral activity of IFN, with inhibition being more prominent with HCV-1bJk NS5A than with M094AJk NS5A. Moreover, the inhibition was significantly weaker in cells expressing a chimeric NS5A that had a short stretch of 49 amino acids (aa 2209-2257), including the ISDR sequence, from M094AJk in the backbone of the HCV-1bJk sequence than in cells expressing the original NS5A from HCV-1bJk. These results suggest an important role for the 49 aa sequence, including the ISDR, in the inhibition of IFN-mediated antiviral activity. On the other hand, only a slight reduction of IFN antiviral activity by HCV-1bJk NS5A was observed when vesicular stomatitis virus was used as a challenge virus, and barely any reduction was observed when Japanese encephalitis virus was used. These results may reflect differential importance of each of the IFN-mediated signalling pathways in conferring resistance against different viruses.

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

A field strain of Sendai virus (SeV) Ohita-M1 (M1) was isolated from an epidemic in an animal laboratory by passaging in mice. A mutant strain, Ohita-MVC11 (MVC11), was then obtained by passaging M1 in rhesus monkey (LLC-MK2) cells. MVC11 was adapted to LLC-MK2 cells and produced 20 times higher levels of infectious virus than M1. This increased production of infectious virus in LLC-MK2 cells was associated with enhanced viral gene expression. However, MVC11 could not replicate efficiently in mouse lung and was not lethal to mice even when inoculated at a titre of 8 x 10(5) cell-infecting units (CIU) per mouse. On the other hand, with an inoculum of only 4 x 10(1) CIU per mouse, corresponding to 1 LD50, M1 replicated well in mouse lung and was highly virulent to mice. Nucleotide and deduced amino acid sequence analyses of the entire genomes of M1 and MVC11 revealed that adaptation to LLC-MK2 cells and the attenuation of mouse pathogenicity of MVC11 were associated with only two amino acid substitutions; one on the C protein (Phe substituted by Ser at position 170) and the other on the RNA polymerase, the L protein (Glu substituted by Ala at position 2050).

Exposure of lentiviral vectors to subneutral pH shifts the tropism from Purkinje cell to Bergmann glia.

Cerebellar Purkinje cells play an important role in cerebellar function; lesions of Purkinje cells result in the disruption of motor coordination and motor learning. Although selective gene delivery to Purkinje cells would be a powerful technique for the study of pathophysiology in the cerebellum, a method for such a delivery has not yet been established. Here we employed human immunodeficiency virus‐derived lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein to transduce Purkinje cells and examined factors that critically affect the viral tropism for Purkinje cells. Viral vectors encoding GFP were generated using different protocols, and were then injected into the mouse cerebellum. At 7 days and 2 months post‐transduction, the relative proportions of transduced Purkinje cells were determined. Lentiviral vectors harvested from a medium of pH 7.2 preferentially transduced Purkinje cells (about half of the transduced cells). In contrast, when the viral vector was harvested from medium of ≤ pH 7.0, only 12–26% of transduced cells were identified as Purkinje cells and 68–77% as Bergmann glia. A similar decrease in the efficiency of transduction for Purkinje cells, depending on the pH of the medium at the viral harvest, was observed in dissociated cell cultures. These results indicate that lentivector tropism for Purkinje cells is extremely sensitive to pH: a subtle decrease in the pH of the medium at the harvest shifts viral tropism strikingly towards Bergmann glia.

Design of a sialylglycopolymer with a chitosan backbone having efficient inhibitory activity against influenza virus infection.

We verified here the inhibitory activity of a sialylglycopolymer prepared from natural products, chitosan and hen egg yolk, against influenza virus infection and estimated the requirements of the molecule for efficient inhibition. The inhibitory activity clearly depended on two factors, the length (the degree of polymerization: DP) of the chitosan backbone and the amount (the degree of substitution: DS) of conjugated sialyloligosaccharide side chain. The inhibitory efficiency increased in accordance with the DP value, with the highest inhibitory activity obtained when the DP was 1430. The inhibition of virus infection reached more than 90% as the DS value increased up to 15.6% when the neighboring sialyloligosaccharide side chains came as close as 4 nm, which was nearly the distance between two receptor-binding pockets in a hemagglutinin trimer. These results demonstrate that the sialylglycopolymer could be an excellent candidate of the safe and efficient anti-influenza drug.

A Tryptophan-Rich Motif in the Human Parainfluenza Virus Type 2 V Protein Is Critical for the Blockade of Toll-Like Receptor 7 (TLR7)- and TLR9-Dependent Signaling

ABSTRACT Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X3-Trp-X9-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

An anti-interferon activity shared by paramyxovirus C proteins: inhibition of Toll-like receptor 7/9-dependent alpha interferon induction.

Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti‐IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN‐α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll‐like receptor (TLR) 7‐ and TLR9‐dependent IFN‐α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti‐IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.

Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction

ABSTRACT Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.

Single amino acid substitution of Sendai virus at the cleavage site of the fusion protein confers trypsin resistance.

Amino acid sequences of fusion (F) proteins of two trypsin-resistant mutants of Sendai virus, TR-2 and TR-5, were deduced from nucleotide analysis of cDNA encoding the F gene and were compared with that of the trypsin-sensitive wild-type Sendai virus. In both mutants, amino acid substitutions were found at residues 116 (Arg----Ile), the cleavage site of the F protein, and 109 (Asn----Asp). Two trypsin-sensitive revertants, TSrev-52 and TSrev-58, derived from TR-5 were both activated by trypsin similarly to the wild-type virus and had a single amino acid reversion from Ile to Arg at residue 116, leaving Asp as before at residue 109. These results indicate that the trypsin sensitivity of Sendai virus can be changed by a single amino acid substitution at the cleavage site of the F protein and a mutation from Arg to Ile is responsible for the acquisition of resistance to trypsin.

Comparative Analysis of Titers of Antibody against Measles Virus in Sera of Vaccinated and Naturally Infected Japanese Individuals of Different Age Groups

ABSTRACT The anti-measles virus (MV) antibody titers in the sera of vaccinees and naturally infected individuals of different age groups were measured to help assess the efficacy of the current MV vaccination in Japan. Neutralizing (NT) antibody titers induced by vaccination were 23.2 times lower than those induced by natural infection and declined significantly by age 20. The once-decreased NT antibody titers of the vaccinees increased 23.6 times during their twenties to titers comparable to those of naturally infected individuals of the same age, implying the possible occurrence of natural infection in vaccinees with decreased anti-MV immunity. Although the current field strains in Japan, types D3 and D5, were reported to differ antigenically from each other and from vaccine strains (type A) to some extent, as demonstrated by different reactivities to monoclonal antibodies, the sera of vaccinees neutralized the two types of field strains and the vaccine strain with the same efficiency. This result suggests that the current vaccine strain would be suitable to elicit protection against types D3 and D5, as long as viral antigenicity is concerned. However, when compared at given hemagglutination inhibition titers, NT antibody titers of vaccinees were 21.1 to 23.2 times lower than those of naturally infected individuals, suggesting a qualitative difference(s) of anti-MV antibodies between the two groups. It should be emphasized that protective immunity induced by the one-dose vaccination currently implemented in Japan may not be strong enough to ensure lifelong immunity. A two-dose vaccination program with higher vaccination coverage needs to be considered in order to effectively control measles in Japan.