Aniko Paul

SUNY: Stony Brook University, Microbiology and Molecular Genetics, Faculty Member

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Papers by Aniko Paul

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Proceedings of the National Academy of Sciences of the United States of America, Oct 10, 2017

Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whos... more Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2(Min), a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2(Min) displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2(Min), 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a...

Properties of synthetic polydeoxyribonucleotide complexes containing adenine and bromouracil

Biochemistry, 1971

The homopolymer polydeoxyribobromouridylic acid (dBrU) has been prepared using Escherichia coli D... more The homopolymer polydeoxyribobromouridylic acid (dBrU) has been prepared using Escherichia coli DNA polymerase I. Two-and three-stranded base-paired homopolymer complexes with polydeoxyriboadenylic acid (dA) have been made, and interactions occurring between the complexes and their component strands have been studied at 0.1 M sodium ion. The thermal stabilities of the homopolymer pair dA. dBrU and the alternating copolymer d(A-BrU). d(A-BrU) have been determined at pH 7 as a function of ionic strength, and at 0.1 M sodium ion as a function of pH. In addition, the thermal stabilities of dA. dT and d(A-T) 9 d(A-T) have been determined at 0.1 M sodium ion as a function of pH. For both d(A-BrU) polymers and d(A-T) polymers, stability is affected by base sequence. The homopolymer pair is more stable than the copolymer pair at all pH values and ionic strengths studied. Other polymer systems show no base sequence effect. Possible sources of the base sequence dependence are discussed. S ynthetic polynucleotides serve as model compounds for paired two-and three-stranded complexes dA. dBrU and naturally occuring nucleic acids and afford the opportunity to dA.dBrUe, and we have compared certain properties of study the reactivity of molecules having a relatively simple, these polymer complexes to those of closely related polymer repeating composition. Polymers can be studied which differ complexes which differ only in the 5-carbon pyrimidine from one another in chemically simple ways. In this study, substituent or the 2'-hydroxy sugar substituent or in base we have prepared the homopolymer dBrU1 and the base-sequencealone. The properties of deoxy polymers containing BrU are of Abbreviations used are: A, BrU, U, T : adenine, bromouracil, uracil, and thymine, respectively. Deoxynucleoside triphosphates, dXTP. Abbreviations for the synthetic polynucleotides and polynucleotide complexes are those of the IUPAC-IUB Commission (Biochemistry 9, 4025 (1970)), for example, dA. dBrUt denotes the three-stranded homopolymer complex composed of one polydeoxyadenylate strand and two polydeoxybromouridylate strands, while d(A-BrU) denotes the deoxy copolymer containing adenine and bromouracil in strictly alternating sequence. Classes of polymer complexes are also referred to. The "deoxy A-BrU polymers" refers to both copolymer and homopolymer complexes. T , = the temperature at the midpoint of an absorbancy transition, pH, = the pH at the midpoint of an absorbancy transition. cp is the molar extinction coefficient relative to phosphorus. The symbol 2 +.

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Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis

The Journal of general virology, 2006

A stem-loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome ha... more A stem-loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a cis-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication in vivo and abolished uridylylation of VPg in vitro. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R(1)NNNAAR(2)NNNNNNR(3), is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an in vitro translation/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered wi...

Structural and functional characterization of the coxsackievirus B3 CRE(2C): Role of CRE(2C) in negative- and positive-strand RNA synthesis

Journal of General Virology

Effects of temperature and lipophilic agents on poliovirus formation and RNA synthesis in a cell-free system

Journal of Virology

The translation and primary processing events of poliovirus polyproteins in HeLa cell extracts we... more The translation and primary processing events of poliovirus polyproteins in HeLa cell extracts were more efficient at 34 degrees C than at 30 or 36 degrees C. The cleavage products of P2 such as 2Apro, 2BC, and 2C appeared early in the reaction before the appearance of the cleavage products of P1 and of 3CDpro, an observation suggesting that P2 was cleaved in cis by 3CDpro. Proteolytic processing of the capsid precursor P1 into VP0, VP1, and VP3 was also more efficient at 34 degrees C than at either 30 or 32 degrees C. Surprisingly, processing of 3CDpro to 3Cpro and 3Dpol was almost completely inhibited at 36 degrees C. The synthesis of virus in the cell extract was greatly enhanced at 34 degrees C over that at 30 or 32 degrees C, whereas incubation at 36 degrees C yielded very little virus. Cerulenin, an inhibitor of lipid synthesis, did not appear to affect virus-specific translation or protein processing, but it almost completely inhibited viral synthesis in vitro. Oleic acid drastically inhibited in vitro translation at 100 microM and in vitro poliovirus synthesis at 25 microM. Addition of HeLa cell smooth membranes partially restored translation but not virus formation. Our observations suggest that in vitro translation, proteolytic processing, and virus formation require intact membranes. Analysis of the in vitro translation products revealed that viral RNA polymerase activity increased linearly during incubation of the translation mixture. RNA polymerase in the crude mixture was inhibited by oleic acid but not by cerulenin. Surprisingly, oleic acid had no direct effect on oligo(U)-primed, poly(A)-dependent poly(U) synthesis catalyzed by purified 3Dpol.

Properties of purified recombinant poliovirus protein 3AB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol

Journal of Biological Chemistry

The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified ... more The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified to near homogeneity. Corresponding to its known association with membranes in poliovirus-infected HeLa cells, 3AB expressed in E. coli was also membrane-associated, and it could be solubilized only in detergent-containing buffers. In soluble form, 3AB was resistant to digestion with the virus-specific proteinases 3Cpro and 3CDpro. However, it was cleaved by these enzymes to 3A and VPg when bound to the bacterial membranes, an observation suggesting that 3AB may deliver the genome-linked protein VPg to the membrane-associated poliovirus replication complex. The specific activity of 3CDpro in processing 3AB was significantly higher than that of 3Cpro. Soluble 3AB was found to stimulate nearly 100-fold poly (A)-dependent, primer-dependent poly(U) synthesis, catalyzed by purified poliovirus RNA polymerase 3Dpol. We propose that 3AB has a dual function in poliovirus genome replication: as a precursor for VPg, and as a co-factor for 3Dpol.

Studies with poliovirus polymerase 3Dpol: Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB

Journal of Biological Chemistry

The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT)15 primer by poliovirus RNA... more The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT)15 primer by poliovirus RNA polymerase 3Dpol (280 ng/ml) is strongly stimulated (50-100 fold) by the addition of purified poliovirus polypeptide 3AB. The synthesis of product continues linearly with time for up to 90 min. The reaction with 3Dpol alone can be reactivated and similarly enhanced by the addition of 3AB at 30 min of incubation. Optimal stimulation is achieved under conditions where the concentration of 3Dpol and of template is low, when the molar ratio of 3AB to 3Dpol is about 100:1 and that of 3AB to poly(A) is about 25:1. In the presence of 3AB, the yield of product made by 3Dpol is much increased but its size is unchanged. From a number of basic proteins and peptides tested, a few were found which also exhibited limited enhancement of polymerase activity. The stimulatory effect of 3AB is probably related to its ability to bind both the template-primer, poly(A).oligo(dT)15, and 3Dpol (Molla, A., Harr...

Interaction of poliovirus polypeptide 3CDpro with the 5′ and 3′ termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding

Journal of Biological Chemistry

Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have ... more Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have been purified from HeLa cells (p50 and p36) which interact with purified 3CDpro but only p36-3CDpro bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segment whose secondary structure resembles a cloverleaf. The identity of these factors was determined by microsequencing tryptic digests of the purified proteins. Host protein p50 is the eukaryotic elongation factor EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpro or 3Dpol. Significantly, the formation of a 3CDpro-cloverleaf complex was also observed in the presence of purified poliovirus polypeptide 3AB, the precursor of VPg. 3AB by itself does not stably bind to the cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. UV cross-linking studies were emp...

Stimulation of poliovirus proteinase 3Cpro-related proteolysis by the genome-linked protein VPg and its precursor 3AB

Journal of Biological Chemistry

Purified recombinant poliovirus polypeptide 3AB interacts with 3CDpro and 3Dpol as shown by coimm... more Purified recombinant poliovirus polypeptide 3AB interacts with 3CDpro and 3Dpol as shown by coimmunoprecipitation with anti-3Dpol antibodies. A consequence of this interaction is an accelerated autoprocessing of 3CDpro to produce 3Cpro and 3Dpol. The activation of 3Dpol polymerase activity by cleavage of 3CDpro, a polypeptide that has no polymerase activity, can be shown by template- and primer-dependent poly(U) synthesis. Anti-VPg antibodies (VPg = 3B) added to HeLa translation extracts programmed with poliovirion RNA inhibit cleavage of 3CDpro whereas addition of purified 3AB or VPg to these translation reactions increases 3CDpro processing. 3AB stimulates also 3Cpro-related proteolysis of 2BC, a poliovirus-specific, nonstructural processing intermediate. In contrast, 3CDpro-specific cleavage of the structural precursor P1 is inhibited by the addition of 3AB as shown by a decrease in the production of VP0 and VP3. These data shed new light on a phenomenon in the regulation of expr...

Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB

RNA

Genome replication of poliovirus, as yet unsolved, involves numerous viral polypeptides that aris... more Genome replication of poliovirus, as yet unsolved, involves numerous viral polypeptides that arise from proteolysis of the viral polyprotein. One of these proteins is 3AB, an RNA-binding protein with multiple functions, that serves also as the precursor for the genome-linked protein VPg (= 3B). Eight clustered charged amino acid-to-alanine mutants in the 3AB coding region of poliovirus were constructed and analyzed, together with three additional single-amino acid exchange mutants in VPg, for viral phenotypes. All mutants expressed severe inhibition in RNA synthesis, but none were temperature sensitive (ts). The 3AB polypeptides of mutants with a lethal phenotype were overexpressed in Escherichia coli, purified to near homogeneity, and studied with respect to four functions: (1) ribonucleoprotein complex formation with 3CDpro and the 5'-terminal cloverleaf of the poliovirus genome; (2) binding to the genomic and negative-sense RNA; (3) stimulation of 3CDpro cleavage; and (4) sti...

A new polio vaccine?

A., which will be glad to provide the latest information on any changes made to the text, plans f... more

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Vaccines should be kept even if polio is wiped out

Nature, Jan 29, 2002

Protein-primed RNA synthesis by purified poliovirus RNA polymerase

Nature, Jan 21, 1998

A small protein, VPg, is covalently linked to the 5' end of the plus-stranded poliovirus geno... more A small protein, VPg, is covalently linked to the 5' end of the plus-stranded poliovirus genomic RNA. Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5' end by the methylated structure that is common to most eukaryotic mRNAs. These discoveries presented two problems. First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5' mRNA end, has solved the first conundrum. Here we describe the resolution of the second problem. VPg is linked to the genomic RNA through the 5'-terminal uridylic acid of the RNA. We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol. Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to pro...

Internal Ribosomal Entry Site Scanning of the Poliovirus Polyprotein: Implications for Proteolytic Processing

Virology, 1998

Based on previous studies of dicistronic polioviruses carrying two internal ribosomal entry sites... more Based on previous studies of dicistronic polioviruses carrying two internal ribosomal entry sites (IRESes), we performed a novel experiment of IRES scanning through a polypeptide by inserting sequentially the IRES of encephalomyocarditis virus into the open reading frame (ORF) of the poliovirus polyprotein at selected 3C pro -specific Q*G cleavage sites. No cytopathic effects were observed after transfection of HeLa cells with any of the dicistronic constructs, and no virus was recovered. In vitro translation of the dicistronic RNA transcripts in HeLa cell-free extracts revealed that multiple defects in the processing of the P2±P3 domain of the polyprotein is the primary reason for the lethal phenotypes. Surprisingly, the interruption of 3C pro -catalyzed cleavages downstream of 2C interfered with the 2A pro -catalyzed, primary cleavage between P1 and P2. In contrast, insertion of a foreign coding sequence (V3 loop of human immunodeficiency virus type 1 gp120) into the ORF of the polyprotein at the 2C±3A junction yielded a viable virus that appeared to be genetically stable over several passages. The results of these experiments, which are generally applicable to analyses of viral polyproteins or multidomain polypeptides, suggest that processing of the P2±P3 domain by 3C±3CD pro is rapid and accurate only in the context of the unperturbed P2-P3 precursor; this is consistent with cleavages occurring in cis. Moreover, an intact 2C±3A precursor is not required for viral proliferation.

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Initiation of protein-primed picornavirus RNA synthesis

Virus research, Jan 12, 2015

Plus strand RNA viruses use different mechanisms to initiate the synthesis of their RNA chains. T... more Plus strand RNA viruses use different mechanisms to initiate the synthesis of their RNA chains. The Picornaviridae family constitutes a large group of plus strand RNA viruses that possess a small terminal protein (VPg) covalently linked to the 5'-end of their genomes. The RNA polymerases of these viruses use VPg as primer for both minus and plus strand RNA synthesis. In the first step of the initiation reaction the RNA polymerase links a UMP to the hydroxyl group of a tyrosine in VPg using as template a cis-replicating element (cre) positioned in different regions of the viral genome. In this review we will summarize what is known about the initiation reaction of protein-primed RNA synthesis by the RNA polymerases of the Picornaviridae. As an example we will use the RNA polymerase of poliovirus, the prototype of Picornaviridae. We will also discuss models of how these nucleotidylylated protein primers might be used, together with viral and cellular replication proteins and other...

Paradoxes of the replication of picornaviral genomes

Virus Research, 1999

A wealth of experimental data on the mechanism of the picornavirus genome replication has accumul... more A wealth of experimental data on the mechanism of the picornavirus genome replication has accumulated. Not infrequently, however, conclusions derived from these data appear to contradict each other. On the one hand, initiation of a complementary RNA strand can be demonstrated to occur in a solution containing only the poliovirus RNA polymerase, VPg, uridine triphosphate, poly(A) template and appropriate ions. On the other hand, convincing experiments suggest that efficient initiation of a viral complementary RNA strand requires complex cis-acting signals on the viral RNA template, additional viral and possibly cellular proteins as well as a membrane-containing environment. On the one hand, there is evidence that the viral RNA, in order to be replicated, should first be translated, but on the other hand, the viral RNA polymerase appears to be unable to overcome the ribosome barrier. Possible solutions for these and several other similar paradoxes are discussed, along with less contradictory results on the properties of the picornaviral replicative proteins. Recent results suggesting that recombination and other rearrangements of the viral RNA genomes may be accomplished not only by the replicative template switching but also by nonreplicative mechanisms are also briefly reviewed.

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Identification of two functionally redundant RNA elements in the coding sequence of poliovirus using computer-generated design

Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2012

Genomes of RNA viruses contain multiple functional RNA elements required for translation or RNA r... more Genomes of RNA viruses contain multiple functional RNA elements required for translation or RNA replication. We use unique approaches to identify functional RNA elements in the coding sequence of poliovirus (PV), a plus strand RNA virus. The general method is to recode large segments of the genome using synonymous codons, such that protein sequences, codon use, and codon pair bias are conserved but the nucleic acid sequence is changed. Such recoding does not affect the growth of PV unless it destroys the sequence/structure of a functional RNA element. Using genetic analyses and a method called "signal location search," we detected two unique functionally redundant RNA elements (α and β), each about 75 nt long and separated by 150 nt, in the 3'-terminal coding sequence of RNA polymerase, 3D(pol). The presence of wild type (WT) α or β was sufficient for the optimal growth of PV, but the alteration of both segments in the same virus yielded very low titers and tiny plaque...

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RNA Signals in Entero- and Rhinovirus Genome Replication

Seminars in Virology, 1997

The VPg-linked, plus-stranded RNA genomes of entero-and rhinoviruses contain very different 5Ј an... more The VPg-linked, plus-stranded RNA genomes of entero-and rhinoviruses contain very different 5Ј and 3Ј terminal regions which harbor signals for RNA replication. The terminal cloverleaf-like structure of the 5Ј-nontranslated region (5ЈNTR) is known to be required for plus-strand RNA synthesis. Genetic evidence suggest that two stem-loop structures and the poly(A) tail of the 3ЈNTR have a function in minus-strand synthesis. All of the nonstructural viral proteins, and possibly also some cellular polypeptides, are believed to be involved in RNA replication. RNA synthesis is initiated on a poly(A) template and involves uridylylation of VPg to yield VPgpU(pU). This precursor is likely to serve as primer for the RNA polymerase 3D pol during both minus-and plus-strand RNA synthesis. 1997 Academic Press

format_quoteKey residues in VPg are crucial for uridylylation, highlighting a precise molecular mechanism guiding poliovirus replication.format_quote

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The entire nucleotide sequence of the genome of human hepatitis A virus (isolate MBB)

Virus Research, 1987

Hepatitis A virus (HAV) is an important human pathogen causing hepatitis, with high incidence in ... more Hepatitis A virus (HAV) is an important human pathogen causing hepatitis, with high incidence in developed as well as in developing countries. No vaccines are available. In order to determine the primary structure of the HAV genome, we have prepared cDNAs from viral RNA and cloned these into plasmid pBR322. These clones were used to determine the entire nucleotide sequence of the HAV RNA by rapid sequencing methods. We have compared this sequence of 7470 bases to known partial sequences, and one complete sequence of HAV RNA which were obtained recently from different strains of HAV. It is hoped that a comparison of sequence data from different isolates will help in the elucidation of the unusual growth pattern of HAV. In addition, it might provide helpful information about the immunological determinants that elicit the antibody response to infection.

format_quoteThe complete genome sequence of HAV strain MBB reveals a polyprotein coding for 2227 triplets, typical for picornaviruses.format_quote

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Studies on the Attenuation Phenotype of Polio Vaccines: Poliovirus RNA Polymerase Derived from Sabin Type 1 Sequence Is Temperature Sensitive in the Uridylylation of VPg

Virology, 2000

Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in t... more Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in the 5Ј NTR and coding sequences of the capsid proteins and viral RNA polymerase, 3D pol . Previous studies have implicated at least two mutations in 3D pol of Sabin 1 vaccine strain [PV1(S)], including a Y73H change, as contributing to these phenotypes. We have used an in vitro assay to test the first step in RNA synthesis, the uridylylation of the terminal protein VPg with 3D pol isolated from PV1(S). Wt and two mutant 3D pol proteins (Y73H, D53N/Y73H) were expressed in Escherichia coli and were purified, and their activities were measured in the synthesis of VPgpU(pU) and of VPg-linked poly(U) at 30 and 39.5°C. Our results show that at 39.5°C the Y73H mutation leads to a defect in the synthesis of VPgpUp(U) and of VPg-poly(U) but not in the elongation of a (dT) 15 primer. The double mutant protein had the same activities as Y73H 3D pol . Using the yeast two-hybrid assay, we detected a reduced interaction between 3D pol molecules carrying either the single or double mutations. Tyrosine-73 maps to the finger domain in the three-dimensional structure of 3D pol . A model will be presented in which a change of Y73 to H73 may interfere with an interaction between two polymerase molecules that, in turn, may interfere with VPg uridylylation. Alternative explanations, however, cannot be excluded at the present time.

format_quoteY73H mutation in 3D pol causes defects in VPg linked precursor synthesis, particularly at elevated temperature.format_quote

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