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C D Atreya

Publications and source records attributed to C D Atreya.

35 records · Page 2Linked to original sources
PubMed1999

Mutations in the retinoblastoma protein-binding LXCXE motif of rubella virus putative replicase affect virus replication.

The rubella virus (RV)-encoded protein NSP90, which contains the retinoblastoma protein (Rb)-binding motif LXCXE, interacts with Rb and RV replication is reduced in cells lacking Rb. Whether the LXCXE motif of RV NSP90 itself is essential for Rb binding and virus replication is not known. Therefore, in the present study, the functional role of this motif was investigated by site-directed mutagenesis in a plasmid from which infectious RV RNA can be produced. Three critical mutations in the motif, two substitutions at the conserved cysteine residue (C --> G and C --> R) and a deletion of the entire motif, were created. A cell-free translated NSP90 C terminus polypeptide containing the deletion did not bind to Rb and a polypeptide carrying the C --> R substitution had barely detectable binding affinity for Rb. Rb binding by the C --> G mutant was reduced significantly compared to that of wild-type protein. Correlating with the binding results, mutant viruses containing the LXRXE and LXGXE motifs had a reduction in replication to < 0.5% and 47% of the wild-type, respectively, while deletion of the motif was found to be lethal. By the first serial passage, replication of the LXRXE-carrying virus had increased from < 0.5% to 2% of the wild-type. Sequencing of the genome of this virus revealed a nucleotide change that altered the motif from LXRXE to LXSXE, which is a known Rb-binding motif in two protein phosphatase subunits. Thus, our results clearly demonstrate that the LXCXE motif is required for efficient RV replication.

Amino Acid Sequence↗
PubMed1998

Implications of calreticulin function in parasite biology.

Calreticulin (CR) is a Ca(2+)-binding, multifunctional protein. The amazing array of CR-associated functions range from intracellular activities in secondary messenger release, protein folding and the modulation of gene expression to potential interactions with host receptors and signaling machinery and recognition by the host immune system. The multifunctional nature of CR may impact upon the ability of cells to recognize extracellular stimuli and coordinate appropriate responses. Identification of CR isolated from parasites and the conservation of its functions suggests that investigations into the contributions of CR to various aspects of parasite biology should be undertaken because it may reveal information regarding parasite interaction with the host and how the parasite may modulate its response to the host.

Journal Article↗
PubMed1998

The rubella virus putative replicase interacts with the retinoblastoma tumor suppressor protein.

In utero fetal infection of rubella virus (RV), a positive-stranded RNA virus, frequently induces birth defects if contracted in the first trimester of pregnancy. The underlying mechanism of RV-induced birth defects is not known. Birth defects are also common in certain DNA viral infections such as human cytomegalovirus (HCMV). During HCMV infection, one of its proteins interacts with a cell growth regulatory protein, the retinoblastoma protein (Rb) and stimulates DNA synthesis which is associated with chromosomal damage and cellular mitotic arrest. These affects have been implicated in HCMV induced teratogenesis. Since RV and HCMV both cause teratogenesis, we postulated that during RV infection, a virus-encoded protein might interact with Rb and affect fetal cell growth. In the present study, we have identified a known Rb-binding motif, L x C x E (LPCAE) in the carboxy-terminal half of the putative replicase (NSP90) of RV and demonstrated that the C-terminal region specifically binds to GST-Rb in vitro. Further, by coimmunoprecipitating NSP90 and Rb using specific antibodies to respective proteins, we have confirmed that NSP90 specifically binds to Rb in vivo as well. In addition, RV replication was shown to be less in null-mutant (Rb-/-) mouse embryonic fibroblast cells than in wild-type (Rb+/+) cells, suggesting a possible physiological role for this interaction. Thus, in facilitating RV replication, binding of NSP90 to Rb potentially alters the cell growth regulatory property of Rb, and this could be one of the initial steps in RV-induced teratogenesis.

Animals↗
PubMed1996

Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity.

Calreticulin has been implicated in multiple cell functions. Recently, we have shown that both human and simian calreticulin are RNA binding proteins and that their binding activity is due to phosphorylation. To demonstrate that the RNA binding property of calreticulin is an intrinsic part of this multi-functional molecule and is evolutionarily conserved, we isolated and characterized the calreticulin gene from the unicellular parasite, Leishmania donovani. Amino acid sequence homology between human and Leishmania calreticulin (L. d. cal) is limited, but like the human homologue, L. d. cal binds Ca+2, can be phosphorylated in vitro and binds certain RNA sequences in a phosphorylation-dependent manner. Unlike human calreticulin, L. d. cal is glycosylated and its binding to endogenous Leishmania RNA is phosphorylation-independent. The binding of L. d. cal to Leishmania RNA suggests that the RNA binding activity of calreticulin has remained evolutionarily conserved.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of the coat protein N-terminal amino acids involved in potyvirus transmission by aphids.

The nature of the amino acids in the N-terminal 'DAGX' motif of the coat protein of tobacco vein mottling virus (TVMV) that have a direct effect on aphid transmissibility of the virion were further defined by site-directed mutagenesis. In the first position of the DAGX motif, Asp or Asn are required for aphid transmissibility. In the second position, the nonpolar residue Ala, but not the nonpolar Gly or Val or the polar Thr and Ser, is compatible with transmissibility. In the third position, the small, neutral, nonpolar Gly appears to be critical; even substitution of Ala, with a minimal side-chain, drastically reduces transmissibility. Although the amino acid following the DAG sequence is not highly conserved among potyviruses, the presence of an acidic Glu or Asp residue at this position in the TVMV coat protein drastically reduces or abolishes aphid transmissibility. An attempt was made to test the hypothesis that trypsin cleavage of the N terminus is involved in the aphid inoculation process by destroying a trypsin cleavage site downstream from the DAGX motif. While the predicted decrease in transmission occurred from infected plants, there was no effect on the transmission of purified virus. Of the 23 mutations in the DAGX region of TVMV reported here and previously, only two, substitutions of Lys and Arg for Asp, had a detectable adverse effect other than on aphid transmissibility. These, and perhaps other, residues near the N terminus function in some phase of the TVMV life cycle, in addition to aphid transmission.

Amino Acid Sequence↗
PubMed1995

The rubella virus RNA binding activity of human calreticulin is localized to the N-terminal domain.

The rubella virus RNA 3' cis-acting element, which is essential for viral negative-strand RNA synthesis, is specifically bound by autophosphorylated calreticulin. Autophosphorylation in recombinant human calreticulin occurs on serine and threonine residues. The RNA-binding and autophosphorylation activities were localized to the N-terminal 180 amino acids. Furthermore, N-terminal deletions revealed that the RNA-binding activity of calreticulin is abrogated upon deletion of the first 10 residues, whereas the autophosphorylation activity resides between amino acids 60 and 180. These results indicate that both the rubella virus RNA-binding and autophosphorylation activities of calreticulin are present in the N-terminal domain.

Animals↗
PubMed1994

Identification of calreticulin as a rubella virus RNA binding protein.

Previously, we observed that sequences at the 3' end of rubella virus (RV) genomic RNA that form a stable stem-loop structure are necessary for initiation of RNA replication. A cytosolic protein found in Vero 76 cells (simian origin) specifically bound to the 3' (+)-stem-loop sequence. In the present study, we have purified the RNA binding protein and identified it as a simian homologue of human calreticulin. The purified calreticulin binds to the RV RNA with specificity similar to the protein present in cytosolic extracts. Human calreticulin antibodies recognize several forms of simian calreticulin, one of which is phosphorylated in vivo. A 2-fold increase in phosphorylation of this form of calreticulin is observed in RV-infected cells. Recombinant human calreticulin can bind RV 3' (+)-stem-loop RNA only after undergoing in vitro phosphorylation. This binding activity is abrogated by pretreatment of phosphorylated recombinant human calreticulin with alkaline phosphatase. The RV RNA was also immunoprecipitated from RV-infected UV-crosslinked Vero 76 cells by using calreticulin antibodies. Our results show that phosphorylated calreticulin is an RNA binding protein and phosphorylation is necessary for this activity. Specific binding of calreticulin to the cis-acting element of RV RNA in vivo suggests a possible role for this interaction in viral replication.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of the helper component-proteinase gene of a potyvirus: effects of amino acid substitutions, deletions, and gene replacement on virulence and aphid transmissibility.

We have previously provided evidence that amino acid substitutions within the N-terminal portion of the helper component-proteinase (HC-Pro) from tobacco vein mottling virus (TVMV), in particular at Lys-307, not only affect the aphid transmission activity of HC-Pro but also have a significant effect on TVMV virulence. In the present study amino acids which differ in their charge properties were substituted at position 307. A highly basic residue was required to retain helper component activity and virulence. Deletion and insertion mutagenesis in the 5' terminus of the HC-Pro gene suggested that this RNA domain may be an essential element for TVMV infectivity. Replacement of the TVMV HC-Pro gene with that from another potyvirus, zucchini yellow mosaic virus, maintained infectivity and aphid transmissibility of the chimeric virus, although symptoms were attenuated. Our results suggest that, in addition to its importance in aphid transmission, the HC-Pro gene may be of general importance in regulating virulence of potyviruses, possibly by interaction of these sequences with the host.

Amino Acid Sequence↗
PubMed1992

Application of genome sequence information in potyvirus taxonomy: an overview.

The application of protein and nucleic acid sequence analysis in evolutionary and phylogenetic studies is well established. Available sequence information for the 5' untranslated region of potyviruses including the fungus-transmitted barley yellow mosaic virus (BaYMV) RNA-1 suggests that a 12-nucleotide conserved sequence, the "potybox" is unique to this group. Various non-structural proteins of potyviruses share considerable "signature" sequence homology across a broad spectrum of unrelated viruses, which makes their value limited to "supergroup" or "superfamily" identity. However, in potyviruses, the coat-protein N-terminal sequences and 3' noncoding regions are variable among viruses, but similar among strains of the same virus. This suggests that these sequences may be an accurate marker of genetic relatedness. Until complete genome sequences from a large number of potyviruses become available and their value in systematics is tested, coat protein and 3' noncoding regions remain as the choice of taxonomic indicators. The reason being, that cloning and sequencing of the coat-protein gene and 3' noncoding regions are less complicated and time consuming and the sequences show significant differences among the virus species within the family Potyviridae.

Animals↗
PubMed1992

Construction of in-frame chimeric plant viral genes by simplified PCR strategies.

The use of polymerase chain reaction (PCR)-based mutagenesis to create chimeric genes is presently not cost-effective because of the size and number of primers as well as the number of PCRs required. We have developed two strategies based on inverse PCR that exploit limited homologies between two DNA molecules to create in-frame chimeric plant viral genes. This report also contains a compilation of information useful for determining possible restriction sites at a given common dipeptide coding motif between any genes of interest.

Amino Acid Sequence↗
PubMed1992

Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants.

Helper component (HC-Pro) is a virus-encoded nonstructural protein required for aphid transmission of potyviruses. In the tobacco vein mottling virus (TVMV) polyprotein, HC-Pro represents a 457 residue polypeptide from amino acid position 257 to 713. Previous sequence comparison studies have suggested that mutations of one or two specific amino acid residues in the HC-Pro protein might result in loss of aphid transmission activity. To test this hypothesis, the initial targets were the residues corresponding to these specific amino acids, a lys to glu change and an ile to val change at amino acid positions 307 and 482, respectively, of the TVMV polyprotein, as well as the combination of the two. Two additional mutations within the HC-Pro representing dipeptide changes thr-ser to ile-asp and thr-ala to leu-glu at amino acid positions (283/284) and (368/369), respectively, were also tested to further define the effects of mutations in this region on helper component activity. The mutations at positions 482 and (368/369) had no effect on aphid transmission activity, while mutation at position 307 completely abolished the activity. Except for the 482 mutation, all the mutations also affected symptomatology and virus accumulation in infected plants. Due to the very low concentrations of HC-Pro in plants infected with the (283/284) mutant, the effect of this dipeptide change on aphid transmission activity could not be assessed. The majority of the tested mutations fall within a putative zinc-finger motif postulated in the cysteine-rich N-terminus of HC-Pro. The possible role of this motif in the potyviruses is further discussed in the light of our present results with TVMV.

Animals↗
PubMed1991

Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus.

Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. The mutation Ala----Thr in this triplet drastically reduced transmission, whereas the mutation Asp----Asn had no effect, and the mutation Asp----Lys consistently reverted to the wild-type residue. The mutation Lys----Glu, in the residue adjacent to the glycine of the triplet, drastically reduced transmission, whereas the mutation Gln----Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence of a glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility.

Amino Acid Sequence↗
PubMed1990

Selection of deletion mutants by polymerase chain reaction.

Polymerase chain reaction (PCR) based DNA amplification has replaced many time-consuming protocols in molecular biology. Here we describe a simple strategy to quickly select deletion mutants based on PCR methodology which then can be confirmed by nucleotide sequencing. A forward PCR primer is designed in such a way to recognize only the wild type sequences in the amplification reaction and thus a negative selection identifies the deletion in the samples.

Base Sequence↗
PubMed1990

A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus.

A nonaphid transmissible (NAT) variant of tobacco vein mottling virus (TVMV) was used to test the hypothesis that the viral coat protein (CP) plays a role in determining aphid transmissibility. Comparison of the nucleotide sequences in the coat protein cistron of an aphid transmissible isolate (TVMV-AT) with that of TVMV-NAT revealed a single nucleotide difference (G----A) at position 8445; this alters a single amino acid residue (G----E) at position 2747. A cDNA fragment representing the CP region of TVMV-NAT was substituted into the CP region of a full-length cDNA clone of TVMV-AT, and transcribed RNA was inoculated to tobacco plants. Aphids were unable to transmit the resultant hybrid virus which had the TVMV-NAT coat protein, although the concentration and infectivity of the hybrid virus in the source plants were similar to those of TVMV-AT. This is the first direct demonstration that a CP mutation affects aphid transmissibility of a potyvirus.

Base Sequence↗
PubMed1989

Localization of multiple TMV encapsidation initiation sites on rbcL gene transcripts.

TMV capsid protein reacts with and encapsidates many of the chloroplast DNA transcripts both in vivo and in vitro to form pseudovirions. We report on the encapsidation initiation reaction with one of the major RNA species found in in vivo formed pseudovirions, the mRNA for the chloroplast-encoded large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL). This mRNA is found to contain at least three sites which are independently capable of reacting with capsid protein oligomers to initiate encapsidation. All three sites react with capsid protein less efficiently in vitro than does the functional viral RNA encapsidation initiation site (ei). The 5' portion of the region that contains the most reactive rbcL site, ei-3, shows significant nucleotide sequence homology with the encapsidation initiation sites of the U1 and Cc strains of TMV and it can assume a folding structure that resembles that postulated for the Cc strain site. A site that acts as a block to rod elongation is present in transcripts from the region just 3' to the segment from which the rbcL mRNA is transcribed, probably close to, or at the transcription termination signal.

Base Sequence↗
PubMed1988

Characterization of the TMV encapsidation initiation site on 18S rRNA.

Tobacco Mosaic Virus capsid protein oligomers react with and encapsidate 18S rRNA from both plant and mammalian sources in vitro. The site (ei) in 18S rRNA which reacts with capsid protein to initiate the packaging reaction has been localized and partially characterized by testing the ability of transcripts from different regions of a cloned Cucurbita pepo rDNA repeat unit to become encapsidated. The 18S rRNA ei is found to react more slowly with capsid protein than does the functional virion ei and to lie within a 43 nucleotide region which starts at position 157 from the 5' terminus of 18S rRNA. When 6 nucleotides are removed from the 5' end, the remaining 37 nucleotide segment is still reactive, but with reduced efficiency. The primary structure of the reactive segment has limited similarity to the virion ei and can be folded into a stem-loop. The first 18 nucleotides of the ei region is highly conserved from an evolutionary standpoint and this may account for the ability of 18S rRNAs from both plant and mammalian sources to be encapsidated.

Capsid↗