Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Mar 1;385(1):245-60.
doi: 10.1016/j.virol.2008.11.041. Epub 2009 Jan 7.

Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor

Zhenghe Li  1 , Judit Pogany, Tadas Panavas, Kai Xu, Anthony M Esposito, Terri Goss Kinzy, Peter D Nagy
Affiliations

Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor

Zhenghe Li et al. Virology. .

Abstract

Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins was confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3' end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Identification of viral RNA-binding proteins by the yeast proteome microarray. (A) Biotinylated TBSV gRNA (noncapped) or (B) BMV RNA1 (capped) probes were used as indicated. Three subarrays are shown at higher magnification to illustrate the binding of host proteins to TBSV (top), to BMV (bottom) or to both RNAs (middle). The array contains variable amounts of yeast proteins (supplied by Invitrogen) which were used to calculate the binding for each protein (see Table 1).
Fig. 2
Fig. 2
Binding of recombinant host proteins to the TBSV (+)repRNA in vitro. Gel mobility shift assay was performed with the 32P-labeled DI-72 (+)repRNA as the probe. The recombinant host proteins purified from E. coli as MBP fusion proteins were tested in three different dilutions: 2.0, 0.6 and 0.2 μg). The purified recombinant MBP (2.0 μg) was used as a negative control.
Fig. 3
Fig. 3
Co-purification of TBSV (+)repRNA with selected yeast proteins from yeast cells. (A) Three TAP-tagged host proteins (expressed from their native promoters and chromosomal locations) were co-expressed with TBSV (+)repRNA in yeast, followed by two-step TAP-affinity purification. Northern blot analysis was used to estimate the amount of co-purified TBSV (+)repRNA present in the purified protein samples. The left side of the panel shows (+)repRNA present after one-step purification, whereas the right side of the panel shows the (+)repRNA present after two-step purification. The ratio of the repRNA present was compared to the control sample obtained from yeast lacking any TAP-tagged protein. (B) The purified proteins were analyzed by SDS-PAGE and silver-staining. The expected full-length proteins are marked with arrowheads. (C) Three GST-tagged host proteins (expressed from GAL1 promoter from expression plasmids) were co-expressed with TBSV (+)repRNA in yeast, followed by one-step GST-affinity purification. Northern blot analysis was used to estimate the amount of co-purified TBSV (+)repRNA present in the purified protein samples. (D) The purified proteins were analyzed by Western blotting using an anti-His antibody. The expected-size proteins are marked with arrowheads.
Fig. 4
Fig. 4
The effect of over-expression of selected yeast proteins on TBSV repRNA accumulation in yeast. (A) The given host protein was over-expressed from the GAL1 promoter prior to launching TBSV repRNA replication from the CUP1 promoter. The accumulation level of the plus-stranded TBSV repRNA was estimated by using Northern blotting with a TBSV-specific probe. The data were normalized based on 18S ribosomal RNA levels. The average effect of each host protein on repRNA accumulation is shown based on 6–8 separate samples. Host proteins in black and gray boxes stimulated and inhibited, respectively, TBSV repRNA accumulation more significantly than over-expression of the APT2 pseudogene (marked with asterisk). The average accumulation of TBSV repRNA in yeast carrying an empty expression vector, shown as wt, was taken as 100% (based on two sets of experiments with total of 12 samples). (B) Western blotting analysis of over-expressed host proteins and p33 and p92 replication proteins in yeast using anti-His antibody. Note that each host proteins carry a 19KDa C-terminal tag.
Fig. 5
Fig. 5
Co-purification of eEF1A with the tombusvirus replicase from yeast. (A) The membrane-enriched fraction of yeast expressing the FLAG/6xHis-tagged p33HF/p92HF (+) or the 6xHis-tagged p33H/p92H (−, lanes 1, 3 and 5) was solubilized and was sequentially purified on Ni-affinity and FLAG-affinity columns. Western-blotting with anti-6xHis antibody detected the presence of p33 and p92pol in the purified replicase complex that is active in an in vitro replication assay (not shown) as demonstrated earlier (Serva and Nagy, 2006). The asterisks indicate p33 homodimers that are partly resistant to denaturing conditions. Yeast cells were subjected to formaldehyde cross-linking (lanes 3–6) prior to lysis, purification and digestion with RNase A (lanes 5–6) (B) Western blotting of the same samples as in panel A with anti-eEF1A antibody. Note that the native eEF1A was expressed from its original promoter and original location on the chromosome. (C) The original amount of eEF1A in solubilized membrane preparations from yeast was detected with anti-eEF1A antibody in total protein samples.
Fig. 6
Fig. 6
Interaction between eEF1A and p33 in the split-ubiquitin two-hybrid assay. The full-length TEF2 sequence was fused to NubG as N-terminal fusion, and the p33 sequence was fused to Cub. The heat shock protein 70 (SSA1) was used as a positive control because it is known to interact with p33 (Serva and Nagy, 2006).
Fig. 7
Fig. 7
Binding of eEF1A to the replication silencer sequence of the TBSV (+)repRNA in vitro. (A) Gel mobility shift assay was performed with the 32P-labeled WT-SL1/SL2/SL3(+) RNA probe. The SL1/SL2/SL3 sequence represents the 86 nt 3′ terminal, highly structured sequence of the TBSV (+)RNA with two known cis-acting elements: the replication silencer element required for the assembly of the viral replicase and gPR that is an essential promoter for initiation of minus-strand synthesis and it is also required for replicase assembly. The mutated/deleted bases are indicated with black boxes. The recombinant eEF1A purified from yeast as GST-fusion protein was tested in three different dilutions: 2.0, 0.8 and 0.4 μg. (B) Template competition assay to test the binding of eEF1A to the viral template. Gel mobility shift assay was performed with the 32P-labeled WT-SL1/SL2/SL3(+) RNA probe and purified eEF1A (as in panel A), whereas the unlabeled competitor RNA was used in 5x, 20x and 100x excess over the labeled one as shown. Template gPR-CCC-UUU(+) has the 3′ terminal three Cs changed to three Us (not shown).
Fig. 8
Fig. 8
The effect of eEF1A on TBSV repRNA accumulation and on the replication proteins. (A) The expression of eEF1A protein was down-regulated from a copper-repressible promoter in TKY616. Replication of the TBSV repRNA was measured by Northern blotting 12 h after initiation of TBSV replication and suppression of eEF1A expression. (B) Accumulation of p33 and (C) eEF1A was estimated by Western blotting using anti-His and anti-eEF1A antibodies, respectively. (D) Reduced accumulation of TBSV repRNA in TKY848 yeast expressing eEF1A(T22S) as the only form of eEF1A 24 h after induction of TBSV repRNA replication. The TBSV repRNA and 18S ribosomal RNA levels were estimated by Northern blotting. RecRNA shows novel recombinant RNAs derived from TBSV repRNA and characterized earlier (Cheng, Serviene, and Nagy, 2006). (E) Accumulation of p92pol and (F) p33 was estimated by Western blotting using anti-His antibody in TKY848 and TKY102 yeast strains. (G) The production of p33 mRNA from an expression plasmid was estimated by Northern blotting using p33 mRNA-specific probe. (H) Translation of p33 in extracts prepared from TKY848 and TKY102 yeast strains. The in vitro translation assay of p33 from an exogenously added mRNA in the presence of 35S methionine was performed with extracts containing comparable amounts of yeast proteins. Samples were taken at various time points as shown. Bottom panel: Translation of MS2-CFP from an exogenously added mRNA was used as a control. The band at the bottom of both gels represents the dye front.
Fig. 9
Fig. 9
Reduced half-life of p33 in TKY848 yeast strain. (A) Translation of mRNAs was stopped in yeast with cyclohexamide, followed by Western blot analysis of p33 levels with anti-His antibody at various time points. Strain TKY102 expressing WT eEF1A was used as a control. (B) Calculation of the half-life of p33 in TKY848 and TKY102. The experiments were repeated and the standard deviation is shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahlquist P, Noueiry AO, Lee WM, Kushner DB, Dye BT. Host factors in positive-strand RNA virus genome replication. J Virol. 2003;77(15):8181–6. - PMC - PubMed
    1. Alfonzo JD, Crother TR, Guetsova ML, Daignan-Fornier B, Taylor MW. APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae. J Bacteriol. 1999;181(1):347–52. - PMC - PubMed
    1. Anand M, Balar B, Ulloque R, Gross SR, Kinzy TG. Domain and nucleotide dependence of the interaction between Saccharomyces cerevisiae translation elongation factors 3 and 1A. J Biol Chem. 2006;281(43):32318–26. - PubMed
    1. Bastin M, Hall TC. Interaction of elongation factor 1 with aminoacylated brome mosaic virus and tRNA’s. J Virol. 1976;20(1):117–22. - PMC - PubMed
    1. Baumstark T, Ahlquist P. The brome mosaic virus RNA3 intergenic replication enhancer folds to mimic a tRNA TpsiC-stem loop and is modified in vivo. Rna. 2001;7(11):1652–70. - PMC - PubMed

Publication types

MeSH terms