Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor

Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to approximately 22 A resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus-receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.     

Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.     

PEI-g-PEG-RGD/small interference RNA polyplex-mediated silencing of vascular endothelial growth factor receptor and its potential as an anti-angiogenic tumor therapeutic strategy

Tumor angiogenesis appears to be achieved by the expression of vascular endothelial growth factor (VEGF) within solid tumors that stimulate host vascular endothelial cell mitogenesis and possibly chemotaxis. VEGF's angiogenic actions are mediated through its high-affinity binding to 2 endothelium-specific receptor tyrosine kinase, Flt-1 (VEGFR1), and Flk-1/KDR (VEGFR2). RNA interference-mediated knockdown of protein expression at the messenger RNA level provides a new therapeutic strategy to overcome various diseases. To achieve high efficacy in RNA interference-mediated therapy, it is critical to develop an efficient delivering system to deliver small interference RNA (siRNA) into tissues or cells site-specifically. We previously reported an angiogenic endothelial cell-targeted polymeric gene carrier, PEI-g-PEG-RGD. This targeted carrier was developed by the conjugation of the ανβ3/ανβ5 integrin-binding RGD peptide (ACDCRGDCFC) to the cationic polymer, branched polyethylenimine, with a hydrophilic polyethylene glycol (PEG) spacer. In this study, we used PEI-g-PEG-RGD to deliver siRNA against VEGFR1 into tumor site. The physicochemical properties of PEI-g-PEG-RGD/siRNA complexes was evaluated. Further, tumor growth profile was also investigated after systemic administration of PEI-g-PEG-RGD/siRNA complexes.     

Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer’s disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer’s disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.     

Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus

Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.     

Cholesterol shuttling is important for RNA replication of coxsackievirus B3 and encephalomyocarditis virus

Picornaviruses are a family of positive‐strand RNA viruses that includes important human and animal pathogens. Upon infection, picornaviruses induce an extensive remodelling of host cell membranes into replication organelles (ROs), which is critical for replication. Membrane lipids and lipid remodelling processes are at the base of RO formation, yet their involvement remains largely obscure. Recently, phosphatidylinositol‐4‐phosphate was the first lipid discovered to be important for the replication of a number of picornaviruses. Here, we investigate the role of the lipid cholesterol in picornavirus replication. We show that two picornaviruses from distinct genera that rely on different host factors for replication, namely the enterovirus coxsackievirus B3 (CVB3) and the cardiovirus encephalomyocarditis virus (EMCV), both recruited cholesterol to their ROs. Although CVB3 and EMCV both required cholesterol for efficient genome replication, the viruses appeared to rely on different cellular cholesterol pools. Treatments that altered the distribution of endosomal cholesterol inhibited replication of both CVB3 and EMCV, showing the importance of endosomal cholesterol shuttling for the replication of these viruses. Summarizing, we here demonstrate the importance of cholesterol homeostasis for efficient replication of CVB3 and EMCV.     

A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.     

Antiviral effects of sophoridine against coxsackievirus B3 and its pharmacokinetics in rats

Coxsackievirus B3 (CVB3) is a major pathogen for acute and chronic viral myocarditis. The aim of this study was to investigate the antiviral effects of sophoridine, an alkaloid extracted from Chinese medicinal herb, Sophora flavescens, against CVB3, and the underlying pharmacokinetics. First, we determined the antiviral effects of sophoridine against CVB3 in in vitro (primarily cultured myocardial cells), in vivo (BALB/c mice) and serum pharmacological experiments. Then, we determined the pharmacokinetic behavior in serum samples of SD rats after oral administration by HPLC. Finally, we determined the effects of sophoridine on the production of cytokines in a murine viral myocarditis model by measuring mRNA expression of some important cytokines in hearts of infected BALB/c mice by RT-PCR. We found that sophoridine exhibited obvious antiviral effects both in vitro and in vivo, and serum samples obtained from rats with oral administration of sophoridine reduced the virus titers in infected myocardial cells. The serum concentration profile correlated closely with antiviral activity profile. Moreover, sophoridine significantly enhanced mRNA expression of IL-10 and IFN-gamma, but decreased TNF-alpha mRNA expression. In conclusion, sophoridine possesses antiviral activities against CVB3, by regulating cytokine expression, and it is likely that sophoridine itself, not its metabolites, is mainly responsible for the antiviral activities. Therefore, sophoridine may represent a potential therapeutic agent for viral myocarditis.     

Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.     

Identification of an RNA silencing suppressor from a plant double-stranded RNA virus

RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf phytoreovirus (RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of beta-glucuronidase in transient assays and enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.     

Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an avirulent strain

Coxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5′ untranslated region (5′UTR) to complete critical viral processes. Here, we directly compared the structure of a 5′UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5′UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5′UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104–184 and repeating the 5′UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5′UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5′UTR lacks structure in the SLII cardiovirulence determinant.     

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro . To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro .     

Replication of semliki forest virus: polyadenylate in plus-strand RNA and polyuridylate in minus-strand RNA.

The 42S RNA from Semliki Forest virus contains a polyadenylate [poly(A)] sequence that is 80 to 90 residues long and is the 3'-terminus of the virion RNA. A poly(A) sequence of the same length was found in the plus strand of the replicative forms (RFs) and replicative intermediates (RIs) isolated 2 h after infection. In addition, both RFs and RIs contained a polyuridylate [poly(U)] sequence. No poly(U) was found in virion RNA, and thus the poly(U) sequence is in minus-strand RNA. The poly(U) from RFs was on the average 60 residues long, whereas that isolated from the RIs was 80 residues long. Poly(U) sequences isolated from RFs and RIs by digestion with RNase T1 contained 5'-phosphorylated pUp and ppUp residues, indicating that the poly(U) sequence was the 5'-terminus of the minus-strand RNA. The poly(U) sequence in RFs or RIs was free to bind to poly(A)-Sepharose only after denaturation of the RNAs, indicating that the poly(U) was hydrogen bonded to the poly(A) at the 3'-terminus of the plus-strand RNA in these molecules. When treated with 0.02 mug of RNase A per ml, both RFs and RIs yielded the same distribution of the three cores, RFI, RFII, and RFIII. The minus-strand RNA of both RFI and RFIII contained a poly(U) sequence. That from RFII did not. It is known that RFI is the double-stranded form of the 42S plus-strand RNA and that RFIII is the experimetnally derived double-stranded form of 26S mRNA. The poly(A) sequences in each are most likely transcribed directly from the poly(U) at the 5'-end of the 42S minus-strand RNA. The 26S mRNA thus represents the nucleotide sequence in that one-third of the 42S plus-strand RNA that includes its 3'-terminus.     

Influenza virus protecting RNA: an effective prophylactic and therapeutic antiviral

Another influenza pandemic is inevitable, and new measures to combat this and seasonal influenza are urgently needed. Here we describe a new concept in antivirals based on a defined, naturally occurring defective influenza virus RNA that has the potential to protect against any influenza A virus in any animal host. This “protecting RNA” (244 RNA) is incorporated into virions which, although noninfectious, deliver the RNA to those cells of the respiratory tract that are naturally targeted by infectious influenza virus. A 120-ng intranasal dose of this 244 protecting virus completely protected mice against a simultaneous challenge of 10 50% lethal doses with influenza A/WSN (H1N1) virus. The 244 virus also protected mice against strong challenge doses of all other subtypes tested (i.e., H2N2, H3N2, and H3N8). This prophylactic activity was maintained in the animal for at least 1 week prior to challenge. The 244 virus was 10- to 100-fold more active than previously characterized defective influenza A viruses, and the protecting activity was confirmed to reside in the 244 RNA molecule by recovering a protecting virus entirely from cloned cDNA. There was a clear therapeutic benefit when the 244 virus was administered 24 to 48 h after a lethal challenge, an effect which has not been previously observed with any defective virus. Protecting virus reduced, but did not abolish, replication of challenge virus in mouse lungs during both prophylactic and therapeutic treatments. Protecting virus is a novel antiviral, having the potential to combat human influenza virus infections, particularly when the infecting strain is not known or is resistant to antiviral drugs.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

The structure of the stemloop D subdomain of coxsackievirus B3 cloverleaf RNA and its interaction with the proteinase 3C

Stemloop D (SLD) of the 5' cloverleaf RNA is the cognate ligand of the coxsackievirus B3 (CVB3) 3C proteinase (3Cpro). Both are indispensable components of the viral replication initiation complex. SLD is a structurally autonomous subunit of the 5' cloverleaf. The SLD structure was solved by NMR spectroscopy to an rms deviation of 0.66 A (all heavy atoms). SLD contains a novel triple pyrimidine mismatch motif with a central Watson-Crick type C:U pair. SLD is capped by an apical uCACGg tetraloop adopting a structure highly similar to stable cUNCGg tetraloops. Binding of CVB3 3Cpro induces changes in NMR spectra for nucleotides adjacent to the triple pyrimidine mismatch and of the tetraloop implying them as sites of specific SLD:3Cpro interaction. The binding of 3Cpro to SLD requires the integrity of those structural elements, strongly suggesting that 3Cpro recognizes a structural motif instead of a specific sequence.