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author:("kosov, Yuri")
1.  3C Protease of Enterovirus 68: Structure-Based Design of Michael Acceptor Inhibitors and Their Broad-Spectrum Antiviral Effects against Picornaviruses 
Journal of Virology  2013;87(8):4339-4351.
We have determined the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3Cpro). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3Cpro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3Cpro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted us to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We synthesized a series of peptidic α,β-unsaturated ethyl esters of increasing length and for each inhibitor candidate, we determined a crystal structure of its complex with the EV68 3Cpro, which served as the basis for the next design round. To exhibit inhibitory activity, compounds must span at least P3 to P1′; the most potent inhibitors comprise P4 to P1′. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concentration [EC50] = 0.5 μM for the best compound). Antiviral activities were determined using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC50s of ≈180 nM against EV71 and ≈60 nM against human rhinovirus 14 in a live virus–cell-based assay. Even the shorter SG75, spanning only P3 to P1′, displayed significant activity (EC50 = 2 to 5 μM) against various rhinoviruses.
doi:10.1128/JVI.01123-12
PMCID: PMC3624371  PMID: 23388726
2.  Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2 
PLoS ONE  2012;7(12):e52065.
Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2Apro or Lpro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2Apro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) Lpro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2Apro and Lpro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified Lpro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.
doi:10.1371/journal.pone.0052065
PMCID: PMC3525551  PMID: 23272212
3.  Functional binding of hexanucleotides to 3C protease of hepatitis A virus 
Nucleic Acids Research  2011;40(7):3042-3055.
Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3Cpro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G5T) as a 3Cpro protease inhibitor. Using 1H NMR spectroscopy, G5T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of 1H, 15N-HSQC experiments the binding site for G5T was located to the C-terminal β-barrel of HAV 3Cpro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G5T-binding site, nor does G5T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
doi:10.1093/nar/gkr1152
PMCID: PMC3326307  PMID: 22156376
4.  The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes 
PLoS Pathogens  2009;5(5):e1000428.
Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
Author Summary
The genome of the SARS coronavirus codes for 16 non-structural proteins that are involved in replicating this huge RNA (approximately 29 kilobases). The roles of many of these in replication (and/or transcription) are unknown. We attempt to derive conclusions concerning the possible functions of these proteins from their three-dimensional structures, which we determine by X-ray crystallography. Non-structural protein 3 contains at least seven different functional modules within its 1922-amino-acid polypeptide chain. One of these is the so-called SARS-unique domain, a stretch of about 338 residues that is completely absent from any other coronavirus. It may thus be responsible for the extraordinarily high pathogenicity of the SARS coronavirus, compared to other viruses of this family. We describe here the three-dimensional structure of the SARS-unique domain and show that it consists of two modules with a known fold, the so-called macrodomain. Furthermore, we demonstrate that these domains bind unusual nucleic-acid structures formed by consecutive guanosine nucleotides, where four strands of nucleic acid are forming a superhelix (so-called G-quadruplexes). SUD may be involved in binding to viral or host-cell RNA bearing this peculiar structure and thereby regulate viral replication or fight the immune response of the infected host cell.
doi:10.1371/journal.ppat.1000428
PMCID: PMC2674928  PMID: 19436709
5.  Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation 
Nucleic Acids Research  2007;35(17):5975-5984.
Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3Cpro). However, PABP is cleaved by HAV 3Cpro in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5′ nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.
doi:10.1093/nar/gkm645
PMCID: PMC2034478  PMID: 17726047
6.  Silencing of Hepatitis A Virus Infection by Small Interfering RNAs 
Journal of Virology  2006;80(11):5599-5610.
Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.
doi:10.1128/JVI.01773-05
PMCID: PMC1472172  PMID: 16699041
7.  A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA 
Nucleic Acids Research  2001;29(12):e57.
To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT–PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3′ elongation of mRNA with guanosine residues, thus immediately preserving the 3′ end of the RNA and creating a unique poly(A)–oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC9T6) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT–PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.
PMCID: PMC55756  PMID: 11410680
8.  Improving Proteolytic Cleavage at the 3A/3B Site of the Hepatitis A Virus Polyprotein Impairs Processing and Particle Formation, and the Impairment Can Be Complemented in trans by 3AB and 3ABC 
Journal of Virology  1999;73(12):9867-9878.
The orchestrated liberation of viral proteins by 3Cpro-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3Cpro from its precursors and to assess the function of the stable processing intermediates 3AB and 3ABC, we studied the effect of cleavage site mutations on hepatitis A virus (HAV) polyprotein processing, particle formation, and replication. Using the recombinant vaccinia virus system, we showed that the normally retarded cleavage at the 3A/3B junction can be improved by altering the amino acid sequence at the scissile bond such that it matches the preferred HAV 3C cleavage sites. In contrast to the processing products of the wild-type polyprotein, 3ABC was no longer detectable in the mutant. VP0 and VP3 were generated less efficiently, implying that processing of the structural protein precursor P1-2A depends on the presence of stable 3ABC and/or 3AB. In addition, cleavage of 2BC was impaired in 3AB/3ABC-deficient mutants. Formation of HAV particles was not affected in mutants with blocked 3A/3B and/or 3B/3C cleavage sites. However, 3ABC-deficient mutants produced small numbers of HAV particles, which could be augmented by coexpressing 3AB or 3ABC. The hydrophobic domain of 3A that has been proposed to mediate membrane anchorage of the replication complex was crucial for restoration of defective particle formation. In vitro transcripts of the various cleavage site mutants were unable to initiate an infectious cycle, and no progeny viruses were obtained even after blind passages. Taken together, the data suggest that accumulation of uncleaved HAV 3AB and/or 3ABC is pivotal for both viral replication and efficient particle formation.
PMCID: PMC113036  PMID: 10559299

Results 1-8 (8)