Identification and characterization of host factor interactions with cis-acting elements of rubella virus RNA

Nakhasi, Hira L.; Singh, Nishi; Pogue, Gregory P.; Cao, Xiqing; Rouault, TA

doi:10.1007/978-3-7091-9326-6_26

Cited by 25 publications

(42 citation statements)

References 12 publications

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“…The phosphorylated form of calreticulin interacts with the 3' (+)-SL RV RNA, a cis-acting element necessary for (-)-strand RNA synthesis (15). Further, after RV infection, the RNA binding activity of this form of calreticulin and its affinity for the 3' end of RV RNA increases temporally with the appearance of (-)-strand RNA synthesis (15). These observations suggest that the specific binding of calreticulin to the RV 3' (+)-SL RNA is important in regulating RV RNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…RV, a member of Togaviridae, possesses a (+)-stranded RNA genome, and little is known about the molecular events occurring during its life cycle (12)(13)(14). We have shown (15) that an inverted repeat sequence of 12 nt, located at the 3' end of genomic RNA and capable of forming a stem-loop (SL) structure, is necessary for initiation of (-)-strand synthesis. In Vero 76 cells, permissive for viral replication, the existence of a host protein (=60 kDa) that interacts specifically with the 3' (+)-SL RNA has been demonstrated (5).…”

mentioning

confidence: 99%

“…In Vero 76 cells, permissive for viral replication, the existence of a host protein (=60 kDa) that interacts specifically with the 3' (+)-SL RNA has been demonstrated (5). An increase in the affinity of host protein binding activity is observed after RV infection (5), coinciding temporally with the appearance of (-)-strand RNA synthesis (15). The binding activity is abrogated upon dephosphorylation of the cytosolic extracts (5), suggesting that phosphorylation of the host proteins may be necessary for this interaction with the RV 3' (+)-SL RNA.…”

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confidence: 99%

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Identification of calreticulin as a rubella virus RNA binding protein.

Singh

Atreya

Nakhasi

1994

Proc. Natl. Acad. Sci. U.S.A.

122

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Previously, we observed that sequences at the 3' end of rubella virus (RV) genomic RNA that form a stable stem-oop structure are necessary for initiation of RNA replication. A cytosolic protein found in Vero 76 cells (simian origin) specifically bound to the 3' (+)-stem4oop 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' (+)-stem4oop 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.Understanding the mechanism of RNA virus replication is of prime significance due to its central role in viral pathogenesis. Despite this importance, few details are known about viral replication processes in eukaryotic systems. Often hostencoded proteins are implicated in viral RNA replication (1-4), potentially contributing enzymatic, structural, or regulatory activities. However, these host factors are poorly characterized, and their functions are not well defined.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Identification of calreticulin as a rubella virus RNA binding protein.

Singh

Atreya

Nakhasi

1994

Proc. Natl. Acad. Sci. U.S.A.

122

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“…The finding of 3'-terminal negative-strand RNA binding proteins in animals and plants suggests that cellular proteins may generally play a role in the initiation of positive-strand RNA synthesis in the replication of positivestrand RNA viruses. The animal cell 3'-terminal negativestrand RNA binding proteins had A4, values of 97K, 79K and 56K (Vero African green monkey cells) [8], 42K, 44K and 52K (chicken embryo fibroblasts) [9], and 50K and 52K (mosquito, Aedes albopictus, cells) [lo]. None of these has yet been purified and further work will be needed to determine if any of them are related to any of the plant 3'-terminal negative-strand RNA binding proteins described here.…”

Section: Discussionmentioning

confidence: 99%

Plant proteins that bind to the 3′‐terminal sequences of the negative‐strand RNA of three diverse positive‐strand RNA plant viruses

1994

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The replication of positive-strand RNA plant viruses, which involves both virus-encoded and plant-encoded proteins, takes place in two stages: synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template and synthesis of progeny positive-strand RNA using the negative-strand RNA as a template. Using gel mobility shift and photochemical crosslinking assays, we have identified three proteins of M, 32K, 50K and 1OOK in extracts of tobacco and spinach leaves that bind to the 3'-terminal sequences of the negative-strand RNA of three diverse positive-strand RNA plant viruses. The 32K protein was purified to near homogeneity by chromatography on columns of Macro-prep high Q, heparin-sepharose, single-stranded DNA cellulose and poly(U)-sepharose. No binding of any of the three proteins to the 3'-termini of the positivestrand RNA or the 5'-termini of the positive-strand RNA or negative-strand RNA of any of the three viruses, or to the 3'-termini of the mRNAs of two chloroplast genes, psbA or petD, could be detected. We propose that 3'-terminal negative-strand RNA binding proteins, which may be widespread in the plant kingdom, could be utilised by at least three different positive-strand RNA plant viruses for the initiation of positive-strand RNA synthesis.

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“…Minimally, the 3Ј 305 nt of the genome was maintained, and therefore this region contains the putative 3Ј CSE. Included in this region is a prominent stemloop [3Ј(ϩ)SL] structure at the end of the SP-ORF, 59 nt from the 3Ј terminal poly(A) tract, shown to interact with at least three cellular proteins, one of which was identified as the autoantigen calreticulin (23,30).Four stem-loop (SL) structures were thermodynamically predicted to exist within the 3Ј terminal 240 nt of the RUB genome (termed SL1 to SL4) (4). Mapping of the elements essential for replication within the 3Ј region from SL2 [the 3Ј (ϩ)SL which is at the 3Ј end of the SP-ORF] through the 3Ј end of the genome using the RUB infectious clone, Robo302, has been done previously (4).…”

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confidence: 99%

Analysis of the 3′ cis -Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

Chen

Frolov

Icenogle

et al. 2004

J Virol

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A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3 cis-acting elements (3 CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3 CSE varied. The 3 region necessary for optimal relative survival consisted of the 3 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3 CSE. Within the 3 CSE, deletions in the ϳ245 nt that overlap the 3 end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the ϳ60-nt 3 untranslated region (UTR) were lethal. None of the 3 CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5 proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3 CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3 CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3 CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.Rubella virus (RUB) is the sole member of the Rubivirus genus within the Togavirus family. The RUB genome consists of a single-stranded, plus-strand RNA molecule 9,762 nucleotides (nt) in length (7) which is 5Ј capped and 3Ј polyadenylated and serves as an mRNA during infection. The genome contains two open reading frames (ORFs): the 5Ј-proximal ORF encodes nonstructural proteins (NS-ORF), including the RNA-dependent RNA polymerase, that are necessary for viral RNA replication, while the 3Ј-proximal ORF encodes the viral structural proteins (SP-ORF), the capsid protein (CP), and two envelope glycoproteins, E1 and E2. The NS-ORF is translated from the genomic RNA, and the SP-ORF is translated from a subgenomic RNA. The untranslated regions (UTRs) within the RUB genome include a 40-nt sequence at the 5Ј end (5Ј UTR), a 118-nt sequence in the intragenic (junction) region between the ORFs (J-UTR), and a 59-nt sequence at the 3Ј end (3Ј UTR). Replication of the RUB genome initiates with the synthesis of a genomic-length minus-strand RNA which is then used as a template for the synthesis of both the plusstrand RNA species. The subgenomic RNA is synthesized from an internal promoter within the J-UTR.cis-Acting elements at the 3Ј end of the RUB genome (3Ј CSE) are proposed to act as promoters for minus-strand R...

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Identification and characterization of host factor interactions with cis-acting elements of rubella virus RNA

Cited by 25 publications

References 12 publications

Identification of calreticulin as a rubella virus RNA binding protein.

Identification of calreticulin as a rubella virus RNA binding protein.

Plant proteins that bind to the 3′‐terminal sequences of the negative‐strand RNA of three diverse positive‐strand RNA plant viruses

Analysis of the 3′ cis -Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

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