scholarly journals Alphavirus RNA Genome Repair and Evolution: Molecular Characterization of Infectious Sindbis Virus Isolates Lacking a Known Conserved Motif at the 3′ End of the Genome

2000 ◽  
Vol 74 (20) ◽  
pp. 9776-9785 ◽  
Author(s):  
Jyothi George ◽  
Ramaswamy Raju
Keyword(s):  
Sindbis Virus ◽  
Repeat Sequence ◽  
Genome Replication ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Long Term Stability ◽  
Nontranslated Region ◽  
Viral Promoters ◽  
Sequence Elements

ABSTRACT The 3′ nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3′CSE). The 3′CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3′CSEs were studied in detail to evaluate the role of the 3′CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3′CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3′ terminal motifs of the SIN genome; iii) the 5′ 1-kb regions of all SIN genomes, regardless of the differences in 3′ terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3′CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3′ terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3′CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.

scholarly journals Requirements at the 3′ End of the Sindbis Virus Genome for Efficient Synthesis of Minus-Strand RNA

2005 ◽  
Vol 79 (8) ◽  
pp. 4630-4639 ◽  
Author(s):  
Richard W. Hardy ◽  
Charles M. Rice
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Genome Replication ◽  
Minus Strand ◽  
Wild Type ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Type 3

ABSTRACT The 3′-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3′ CSE) immediately preceding the 3′-poly(A) tail. The 3′ CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3′ CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3′ CSE, (iii) deletion or substitution of the 3′ 13 nucleotides of the 3′ CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3′ sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3′ sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3′ component of the minus-strand promoter is the wild-type 3′ CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.

scholarly journals In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

1999 ◽  
Vol 73 (3) ◽  
pp. 2410-2419 ◽  
Author(s):  
Ramaswamy Raju ◽  
Mustapha Hajjou ◽  
Kristie R. Hill ◽  
Vandana Botta ◽  
Sisir Botta
Keyword(s):  
Sindbis Virus ◽  
De Novo ◽  
Genome Replication ◽  
Sequence Motifs ◽  
Rna Sequences ◽  
Cytoplasmic Polyadenylation ◽  
Nontranslated Region ◽  
Rna Genome ◽  
Negative Sense

ABSTRACT Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

scholarly journals Both conserved signals on mammalian histone pre-mRNAs associate with small nuclear ribonucleoproteins during 3' end formation in vitro.

1987 ◽  
Vol 7 (5) ◽  
pp. 1663-1672 ◽  
Author(s):  
K L Mowry ◽  
J A Steitz
Keyword(s):  
Histone H3 ◽  
Nuclear Extract ◽  
Sequence Element ◽  
Conserved Sequence ◽  
In Vitro Processing ◽  
Sequence Elements ◽  
Rna Fragments ◽  
Sm Protein ◽  
Small Nuclear Ribonucleoproteins

Pre-mRNA substrates containing sequences from human and mouse histone genes are accurately processed in a HeLa cell nuclear extract to generate mature 3' termini. When in vitro processing reactions containing either human histone H3 or mouse histone H3 transcripts are treated with RNase T1 and probed with antibodies specific for the Sm protein determinants or for the trimethylguanosine cap structure unique to the U RNAs present in small nuclear ribonucleoproteins, RNA fragments that encompass the site of 3' end formation on the pre-mRNA transcript are selectively recovered. Several different interactions are detected: at time zero, the protected region contains the upstream conserved hairpin loop structure; at later times during the reaction, protection extends beyond the site of 3' end formation to include the downstream conserved sequence element and the 5' cap of the transcript is bound as well. Possible interactions between Sm small nuclear ribonucleoproteins and these conserved sequence elements in histone pre-mRNAs are discussed.

scholarly journals Changes of the Secondary Structure of the 5′ End of the Sindbis Virus Genome Inhibit Virus Growth in Mosquito Cells and Lead to Accumulation of Adaptive Mutations

2004 ◽  
Vol 78 (10) ◽  
pp. 4953-4964 ◽  
Author(s):  
Rafik Fayzulin ◽  
Ilya Frolov
Keyword(s):  
Virus Replication ◽  
Sindbis Virus ◽  
Specific Protein ◽  
Complex Nature ◽  
Sequence Element ◽  
Virus Growth ◽  
Adaptive Mutations ◽  
Conserved Sequence ◽  
Mosquito Cells ◽  
In Cells

ABSTRACT Both the 5′ end of the Sindbis virus (SIN) genome and its complement in the 3′ end of the minus-strand RNA synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (RdRp). In addition to the 5′ untranslated region (UTR), which was shown to be critical for the initiation of replication, another 5′ sequence element, the 51-nucleotide (nt) conserved sequence element (CSE), was postulated to be important for virus replication. It is located in the nsP1-encoding sequence and is highly conserved among all members of the Alphavirus genus. Studies with viruses containing clustered mutations in this sequence demonstrated that this RNA element is dispensable for SIN replication in cells of vertebrate origin, but its integrity can enhance the replication of SIN-specific RNAs. However, we showed that the same mutations had a deleterious effect on virus replication in mosquito cells. SIN with a mutated 51-nt CSE rapidly accumulated adaptive mutations in the nonstructural proteins nsP2 and nsP3 and the 5′ UTR. These mutations functioned synergistically in a cell-specific manner and had a stimulatory effect only on the replication of viruses with a mutated 51-nt CSE. Taken together, the results suggest the complex nature of interactions between nsP2, nsP3, the 5′ UTR, and host-specific protein factors binding to the 51-nt CSE and involved in RdRp formation. The data also demonstrate an outstanding potential of alphaviruses for adaptation. Within one passage, SIN can adapt to replication in cells of a vertebrate or invertebrate origin.

Both conserved signals on mammalian histone pre-mRNAs associate with small nuclear ribonucleoproteins during 3' end formation in vitro

1987 ◽  
Vol 7 (5) ◽  
pp. 1663-1672
Author(s):  
K L Mowry ◽  
J A Steitz
Keyword(s):  
Histone H3 ◽  
Nuclear Extract ◽  
Sequence Element ◽  
Conserved Sequence ◽  
In Vitro Processing ◽  
Sequence Elements ◽  
Rna Fragments ◽  
Sm Protein ◽  
Small Nuclear Ribonucleoproteins

Pre-mRNA substrates containing sequences from human and mouse histone genes are accurately processed in a HeLa cell nuclear extract to generate mature 3' termini. When in vitro processing reactions containing either human histone H3 or mouse histone H3 transcripts are treated with RNase T1 and probed with antibodies specific for the Sm protein determinants or for the trimethylguanosine cap structure unique to the U RNAs present in small nuclear ribonucleoproteins, RNA fragments that encompass the site of 3' end formation on the pre-mRNA transcript are selectively recovered. Several different interactions are detected: at time zero, the protected region contains the upstream conserved hairpin loop structure; at later times during the reaction, protection extends beyond the site of 3' end formation to include the downstream conserved sequence element and the 5' cap of the transcript is bound as well. Possible interactions between Sm small nuclear ribonucleoproteins and these conserved sequence elements in histone pre-mRNAs are discussed.

Ribosomal protein S19 expression during erythroid differentiation

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 318-324 ◽  
Author(s):  
Lydie Da Costa ◽  
Goutham Narla ◽  
Thiébaut-Noel Willig ◽  
Luanne L. Peters ◽  
Marilyn Parra ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Regulatory Element ◽  
Erythroid Differentiation ◽  
Gene Encoding ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Diamond Blackfan Anemia ◽  
Sequence Elements ◽  
Ribosomal Protein S19 ◽  
Terminal Erythroid Differentiation

Abstract The gene encoding ribosomal protein S19 (RPS19) has been shown to be mutated in 25% of the patients affected by Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. As the role of RPS19 in erythropoiesis is still to be defined, we performed studies on RPS19 expression during terminal erythroid differentiation. Comparative analysis of the genomic sequences of human and mouse RPS19genes enabled the identification of 4 conserved sequence elements in the 5′ region. Characterization of transcriptional elements allowed the identification of the promoter in the human RPS19 gene and the localization of a strong regulatory element in the third conserved sequence element. By Northern blot and Western blot analyses of murine splenic erythroblasts infected with the anemia-inducing strain Friend virus (FAV cells), RPS19 mRNA and protein expression were shown to decrease during terminal erythroid differentiation. We anticipate that these findings will contribute to further development of our understanding of the contribution of RPS19 to erythropoiesis.

Stability and Change in Emotional Intelligence: Exploring the Transition to Young Adulthood

2005 ◽  
Vol 26 (2) ◽  
pp. 100-106 ◽  
Author(s):  
James D.A. Parker ◽  
Donald H. Saklofske ◽  
Laura M. Wood ◽  
Jennifer M. Eastabrook ◽  
Robyn N. Taylor
Keyword(s):  
High School ◽  
Emotional Intelligence ◽  
Postsecondary Education ◽  
Life Transition ◽  
Full Time ◽  
Major Life ◽  
Long Term Stability ◽  
Time Period ◽  
Transition From High School

Abstract. The concept of emotional intelligence (EI) has attracted growing interest from researchers working in various fields. The present study examined the long-term stability (32 months) of EI-related abilities over the course of a major life transition (the transition from high school to university). During the first week of full-time study, a large group of undergraduates completed the EQ-i:Short; 32 months later a random subset of these students (N = 238), who had started their postsecondary education within 24 months of graduating from high school, completed the measures for a second time. The study found EI scores to be relatively stable over the 32-month time period. EI scores were also found to be significantly higher at Time 2; the overall pattern of change in EI-levels was more than can be attributed to the increased age of the participants.

Multi-Center Calibration of the Second Reference Material for Thromboplastin, Rabbit, Plain, Coded CRM 149R

1991 ◽  
Vol 65 (03) ◽  
pp. 263-267 ◽  
Author(s):  
A M H P van den Besselaar ◽  
R M Bertina
Keyword(s):  
Reference Material ◽  
Standard Error ◽  
Standard Errors ◽  
Sensitivity Index ◽  
Long Term Stability ◽  
Significant Bias ◽  
The Mean ◽  
The Relationship ◽  
International Sensitivity Index

SummaryIn a collaborative trial of eleven laboratories which was performed mainly within the framework of the European Community Bureau of Reference (BCR), a second reference material for thromboplastin, rabbit, plain, was calibrated against its predecessor RBT/79. This second reference material (coded CRM 149R) has a mean International Sensitivity Index (ISI) of 1.343 with a standard error of the mean of 0.035. The standard error of the ISI was determined by combination of the standard errors of the ISI of RBT/79 and the slope of the calibration line in this trial.The BCR reference material for thromboplastin, human, plain (coded BCT/099) was also included in this trial for assessment of the long-term stability of the relationship with RBT/79. The results indicated that this relationship has not changed over a period of 8 years. The interlaboratory variation of the slope of the relationship between CRM 149R and RBT/79 was significantly lower than the variation of the slope of the relationship between BCT/099 and RBT/79. In addition to the manual technique, a semi-automatic coagulometer according to Schnitger & Gross was used to determine prothrombin times with CRM 149R. The mean ISI of CRM 149R was not affected by replacement of the manual technique by this particular coagulometer.Two lyophilized plasmas were included in this trial. The mean slope of relationship between RBT/79 and CRM 149R based on the two lyophilized plasmas was the same as the corresponding slope based on fresh plasmas. Tlowever, the mean slope of relationship between RBT/79 and BCT/099 based on the two lyophilized plasmas was 4.9% higher than the mean slope based on fresh plasmas. Thus, the use of these lyophilized plasmas induced a small but significant bias in the slope of relationship between these thromboplastins of different species.

Sign in / Sign up

Export Citation Format

Share Document