Ryuichi Sugiyama

Structure, interaction and real‐time monitoring of the enzymatic reaction of wild‐type APOBEC3G

Human APOBEC3G exhibits anti‐human immunodeficiency virus‐1 (HIV‐1) activity by deaminating cytidines of the minus strand of HIV‐1. Here, we report a solution structure of the C‐terminal deaminase domain of wild‐type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real‐time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3′ → 5′ order. Virus infectivity factor (Vif) of HIV‐1 counteracts the anti‐HIV‐1 activity of APOBEC3G. The structure of the N‐terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species‐specific sensitivity of APOBEC3G to Vif action.

ZNF10 inhibits HIV-1 LTR activity through interaction with NF-κB and Sp1 binding motifs

Kruppel‐associated box‐containing zinc finger (KRAB‐ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB‐ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV‐1 gene expression. We identified 5 KRAB‐ZFPs that suppressed ⩾50% of HIV‐1 LTR. Of the 5 identified KRAB‐ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV‐1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1‐gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense.

The interaction between human initiation factor eIF3 subunit c and heat-shock protein 90: A necessary factor for translation mediated by the hepatitis C virus internal ribosome entry site

Heat-shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of various transcription factors and protein kinases in signal transduction. The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives translation by directly recruiting the 40S ribosomal subunits that bind to eukaryotic initiation factor 3 (eIF3). Our data indicate that Hsp90 binds indirectly to eIF3 subunit c by interacting with it through the HCV IRES RNA, and the functional consequence of this Hsp90-eIF3c-HCV-IRES RNA interaction is the prevention of ubiquitination and the proteasome-dependent degradation of eIF3c. Hsp90 activity interference by Hsp90 inhibitors appears to be the result of the dissociation of eIF3c from Hsp90 in the presence of HCV IRES RNA and the resultant induction of the degradation of the free forms of eIF3c. Moreover, the interaction between Hsp90 and eIF3c is dependent on HCV IRES RNA binding. Furthermore, we demonstrate, by knockdown of eIF3c, that the silencing of eIF3c results in inhibitory effects on translation of HCV-derived RNA but does not affect cap-dependent translation. These results indicate that the interaction between Hsp90 and eIF3c may play an important role in HCV IRES-mediated translation.

Heat Shock Protein 70 Inhibits HIV-1 Vif-mediated Ubiquitination and Degradation of APOBEC3G *

The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner.

Effects of resistance-associated NS5A mutations in hepatitis C virus on viral production and susceptibility to antiviral reagents

Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have potent anti-HCV effects but may provoke resistance-associated variants (RAVs). In this study, we assessed the characteristics of these RAVs and explored efficacious anti-HCV reagents using recombinant HCV with NS5A from a genotype 1b strain. We replaced the NS5A of JFH1 with that of Con1 (JFH1/5ACon1) and introduced known NS5A inhibitor resistance mutations (L31M, L31V, L31I and Y93H) individually or in combination. Susceptibilities against anti-HCV reagents were also investigated. RAVs with Y93H exhibited high extracellular core antigen levels and infectivity titers. Variants with any single mutation showed mild to moderate resistance against NS5A inhibitors, whereas variants with double mutations at both L31 and Y93 showed severe resistance. The variants with mutations exhibited similar levels of susceptibility to interferon (IFN)-α, IFN-λ1, IFN-λ3 and Ribavirin. Variants with the Y93H mutation were more sensitive to protease inhibitors compared with JFH1/5ACon1. In conclusion, the in vitro analysis indicated that the Y93H mutation enhanced infectious virus production, suggesting advantages in the propagation of RAVs with this mutation. However, these RAVs were susceptible to protease inhibitors. Thus, a therapeutic regimen that includes these reagents is a promising means to eradicate these RAVs.

RNA Interference Targeted to the Conserved Dimerization Initiation Site (DIS) of HIV-1 Restricts Virus Escape Mutation

Abstract Short hairpin RNAs (shRNA) targeting viral or cellular genes can effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication. This inhibition, however, may induce mutations in the targeted gene, leading to rapid escape from the shRNA-induced inhibition. We generated a lymphoid cell line that stably expressed a 19-bp shRNA targeting a well-conserved dimerization initiation site (DIS) of HIV-1, which strongly inhibited viral replication, thereby delaying virus escape. Furthermore, treatment of HIV-1 infection with DIS- and vif-shRNA combination therapy resulted in superior anti-viral responses compared to vif-shRNA monotherapy. Continuous challenge with HIV-1, however, generated virus mutants that could overcome the RNA interference restriction. Such anti-genes may be promising tools for HIV-1 gene therapy for HIV/acquired immunodeficiency syndrome.

Amino Acid Polymorphisms in Hepatitis C Virus Core Affect Infectious Virus Production and Major Histocompatibility Complex Class I Molecule Expression.

Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells, and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production, and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV.

HIV-1 RT-dependent DNAzyme expression inhibits HIV-1 replication without the emergence of escape viruses

DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tRMDtRL, or one of two truncated tRNAs (Lys-3), tRMDΔARMtRL or tRMD3′-endtRL. Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.

A new role for PGA1 in inhibiting hepatitis C virus-IRES-mediated translation by targeting viral translation factors.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.

Induction of heat-shock protein 70 by prostaglandin A1 inhibits HIV-1 Vif-mediated degradation of APOBEC3G

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A₁ (PGA₁) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin-proteasome pathway, we examined the effects of PGA₁ on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA₁ blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA₁-treated 293T cells. The induction of HSP70 synthesis by PGA₁ significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA₁. Thus, we showed that PGA₁ inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.