Interaction between the Cellular Protein eEF1A and the 3′-Terminal Stem-Loop of West Nile Virus Genomic RNA Facilitates Viral Minus-Strand RNA Synthesis

ABSTRACT RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3′(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3′(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3′ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3′ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3′ SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3′ end of the genome and the RC. eEF1A bound with similar efficiencies to the 3′-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Download Full-text

Mutation of Mapped TIA-1/TIAR Binding Sites in the 3′ Terminal Stem-Loop of West Nile Virus Minus-Strand RNA in an Infectious Clone Negatively Affects Genomic RNA Amplification

Journal of Virology ◽

10.1128/jvi.00991-08 ◽

2008 ◽

Vol 82 (21) ◽

pp. 10657-10670 ◽

Cited By ~ 39

Author(s):

Mohamed M. Emara ◽

Hsuan Liu ◽

William G. Davis ◽

Margo A. Brinton

Keyword(s):

West Nile Virus ◽

T Cell ◽

Protein Binding ◽

Infectious Clone ◽

Virus Production ◽

Minus Strand ◽

Genomic Rna ◽

Stem Loop ◽

Intracellular Antigen

ABSTRACT Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3′ minus-strand stem-loop [WNV3′(−)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3′(−)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3′(−)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3′ (−)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3′(−)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Download Full-text

Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Journal of Virology ◽

10.1128/jvi.74.24.11671-11680.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11671-11680 ◽

Cited By ~ 49

Author(s):

T. A. M. Osman ◽

C. L. Hemenway ◽

K. W. Buck

Keyword(s):

Rna Polymerase ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Central Core ◽

Minus Strand ◽

Stem Loop ◽

Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

Download Full-text

Cell Proteins TIA-1 and TIAR Interact with the 3′ Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication

Journal of Virology ◽

10.1128/jvi.76.23.11989-12000.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 11989-12000 ◽

Cited By ~ 139

Author(s):

W. Li ◽

Y. Li ◽

N. Kedersha ◽

P. Anderson ◽

M. Emara ◽

...

Keyword(s):

West Nile Virus ◽

Rna Binding ◽

Peptide Sequencing ◽

Viral Rna ◽

Cell Protein ◽

Affinity Column ◽

Rna Recognition Motif ◽

The West ◽

West Nile ◽

Stem Loop

ABSTRACT It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3′-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3′(−) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3′(−) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3′(−) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3′(−) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (Kd ) for the interaction between TIAR RRM2 and the WNV 3′(−) SL RNA was 1.5 × 10−8, while that for TIA-1 RRM2 was 1.12 × 10−7. WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

Download Full-text

Faculty Opinions recommendation of Cell proteins TIA-1 and TIAR interact with the 3' stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008146.152807 ◽

2002 ◽

Author(s):

Lee Gehrke

Keyword(s):

West Nile Virus ◽

Virus Replication ◽

Minus Strand ◽

The West ◽

West Nile ◽

Stem Loop

Download Full-text

Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

Journal of Virology ◽

10.1128/jvi.79.14.9046-9053.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 9046-9053 ◽

Cited By ~ 16

Author(s):

Jen-Wen Lin ◽

Hsiao-Ning Chiu ◽

I-Hsuan Chen ◽

Tzu-Chi Chen ◽

Yau-Heiu Hsu ◽

...

Keyword(s):

Mosaic Virus ◽

Rna Synthesis ◽

Viral Rna ◽

Minus Strand ◽

Internal Deletion ◽

Stem Loop ◽

Cis Acting ◽

Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

Download Full-text

Genetic Interactions among the West Nile Virus Methyltransferase, the RNA-Dependent RNA Polymerase, and the 5′ Stem-Loop of Genomic RNA

Journal of Virology ◽

10.1128/jvi.00654-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 7047-7058 ◽

Cited By ~ 46

Author(s):

Bo Zhang ◽

Hongping Dong ◽

Yangsheng Zhou ◽

Pei-Yong Shi

Keyword(s):

West Nile Virus ◽

Rna Polymerase ◽

Viral Replication ◽

Genetic Interactions ◽

Genome Length ◽

Genomic Rna ◽

Rna Dependent Rna Polymerase ◽

West Nile ◽

Stem Loop ◽

Compensatory Mutations

ABSTRACT Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m7GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5′-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5′-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5′ stem-loop of the genomic RNA.

Download Full-text

Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts

Journal of Virology ◽

10.1128/jvi.77.10.5703-5711.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5703-5711 ◽

Cited By ~ 15

Author(s):

K. Sivakumaran ◽

M. Hema ◽

C. Cheng Kao

Keyword(s):

Mosaic Virus ◽

Rna Synthesis ◽

Previous Analysis ◽

Brome Mosaic Virus ◽

Minus Strand ◽

Stem Loop ◽

Virus Rna ◽

Different Levels

ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

Download Full-text