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
. 2006 Sep;80(17):8459-68.
doi: 10.1128/JVI.00545-06.

Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound

Masaki Nishikiori  1 , Koji Dohi, Masashi Mori, Tetsuo Meshi, Satoshi Naito, Masayuki Ishikawa
Affiliations

Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound

Masaki Nishikiori et al. J Virol. 2006 Sep.

Abstract

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
The estradiol-inducible ToMV infection system. (A) Schematic representation of the estradiol-inducible infection system for a ToMV derivative that carries the ER-targeted GFP (erGFP) gene. A modified ToMV cDNA was placed between an estradiol-inducible promoter and a ribozyme sequence (Rz) followed by a polyadenylation signal (Ter). Upon estradiol induction, a modified ToMV RNA is produced via transcription by cellular RNA polymerase II followed by ribozyme cleavage. This modified ToMV RNA is translated to produce the 130K and 180K replication proteins and subjected to replication. A subgenomic mRNA encoding erGFP is synthesized during replication. (B to E) Estradiol-induced ToMV infection in transgenic BY-2 cells. Nomarski and GFP fluorescence images are superimposed. (B) Uninduced cells; (C) induced cells (2 days after induction); (D) protoplasts prepared from induced cells; (E) evacuolated protoplasts prepared from induced cells. Bars, 25 μm.
FIG. 2.
FIG. 2.
Effects of evacuolation on ToMV RdRp activity in extracts of infected protoplasts. Equal volumes of transgenic BY-2 protoplast extracts with (+) or without (−) estradiol induction or evacuolation were analyzed. (A) RdRp activity. Cell extracts were assayed for RdRp activity without exogenous RNA template in the presence of [α-32P]CTP. Total RNA was purified from the reaction mixtures and separated by 7 M urea-2.4% polyacrylamide gel electrophoresis, and 32P-labeled bands were visualized using a Fuji BAS 2500 imaging analyzer (left panel). For comparison, transgenic BY-2 cells were cultured in the presence (+) or absence (−) of estradiol for 24 h, and protoplasts were formed and cultured in a medium containing [3H]uridine and actinomycin D (100 μg/ml) for 2 h. Labeled RNA was purified and separated as described above and visualized by fluorography (right panel) (18). The positions corresponding to ToMV genomic ssRNA [G (ss)], RF [RF (ds)], and the GFP subgenomic ssRNA [Sg (ss)] are indicated at right. (B) Strand specificity of RdRp products. 32P-labeled RdRp reaction products from panel A were subjected to RNase protection assays (RPA) using unlabeled P1M and P7P probes (14) to detect ToMV (+)RNAs and (−)RNAs, respectively. (C) Detection of ToMV (−)RNA by Northern hybridization with a 32P-labeled P7P RNA probe (14). (D) Detection of ToMV 130K and 180K proteins and host proteins Sec61, TOM1, and TOM2A by Western blot analysis. The 100,000 × g membrane pellet fraction of each extract was used to detect Sec61, TOM1, and TOM2A. The band marked by the asterisk has not been characterized.
FIG. 3.
FIG. 3.
Fractionation of the ToMV RdRp activity and replication proteins by centrifugation. iBYL was subjected to a centrifugation at 15,000 × g for 15 min to obtain the supernatant (S15) and pellet (P15) fractions. Lanes S15, P15, and Total represent equal volumes of iBYL (+). An uninfected, evacuolated BY-2 protoplast extract was concurrently analyzed (−). (A) RdRp activity in the absence of exogenous template. (B) RdRp activity in the presence of exogenous ToMV RNA template (1 μg of ToMV RNA in a 50-μl reaction mixture). The RdRp assay was performed, and the products were analyzed as described in the legend to Fig. 2A. For abbreviations, see the legend to Fig. 2A. (C) Strand specificity of RdRp products. The 32P-labeled RdRp reaction products shown in panel B (reaction in the presence of exogenous ToMV RNA template) were analyzed by RNase protection assays (RPA) as described in the legend to Fig. 2B. (D) Detection of ToMV (−)RNA by Northern hybridization with a 32P-labeled P7P RNA probe (14). (E) Detection of ToMV 130K and 180K proteins and host membrane protein Sec61 by Western blot analysis.
FIG. 4.
FIG. 4.
Membrane flotation analysis of ToMV RdRp activity and replication proteins. (A) Schematic representation of the membrane flotation procedure. The iBYL P15 fraction was treated with either TR buffer (mock treatment) (B), 1 M NaCl (C), 0.1 M Na2CO3 (D), or 1% Triton X-100 (E) and then fractionated into top (T), middle (M), and bottom (B) fractions of equal volume. Equal volumes of fractionated samples were then subjected to Western blot analysis for detection of ToMV 130K and 180K proteins and the host proteins Sar1 (a peripheral membrane protein), Sec61 (an integral membrane protein), and TOM1 (an integral membrane protein); Northern hybridization for detection of ToMV (−)RNA; and the RdRp assay. RdRp activity was assayed in the absence of exogenous template RNA. The experimental conditions and abbreviations are described in the legends to Fig. 2D, C, and A for Western, Northern, and RdRp analyses, respectively.
FIG. 5.
FIG. 5.
MNase sensitivity of (−)RNA. The iBYL membranes were suspended in TR buffer containing 2 mM calcium acetate and incubated in the absence (Triton − or MNase−) or presence (Triton + or Mnase 1 or 5) of 1 U/μl or 5 U/μl MNase and 1% Triton X-100 at 30°C for 30 min. After the reaction, total RNA was purified by phenol extraction in the presence of 0.2% SDS. ToMV (−)RNA was detected by Northern hybridization with a 32P-labeled P7P RNA probe (14).
FIG. 6.
FIG. 6.
Effects of detergents on ToMV RdRp activity and the state of the replication proteins. (A) Effect of detergents on ToMV RdRp activity. The iBYL membranes were incubated in the absence or presence of 1% (wt/vol) of the indicated detergents at 15°C for 20 min and then subjected to the RdRp assay. The RdRp assay was performed and the products were analyzed as described in the legend to Fig. 3A and B. For abbreviations, see the legend to Fig. 2A. (B) Western blot analysis of viral and host proteins. The iBYL membranes were treated with the indicated detergents and then subjected to centrifugation at 15,000 × g for 15 min. The resulting supernatants (S15) were used for Western blot analysis. As a control, mock-treated iBYL membranes were centrifuged as above, the resulting pellet (P15) was resuspended with TR buffer to the same final volume as the supernatant fraction, and both fractions were analyzed. ToMV replication proteins, an integral membrane protein Sec61, and ER lumen-localized GFP (erGFP) were detected.
FIG. 7.
FIG. 7.
Sucrose gradient sedimentation analysis of ToMV replication proteins, (−)RNA, and RdRp activity. The iBYL S15 fraction (A to C) or iBYL membranes (D to F) were mock treated (A, D) or treated with 1% Triton X-100 (B, E) or 1% LPC (C, F) at 15°C for 20 min. Samples were then loaded onto a 10 to 50% continuous sucrose gradient and centrifuged. Eight fractions (numbered 1 to 8, from top to bottom of a gradient) were collected and subjected to RdRp assays with or without exogenous ToMV RNA template (0.5 μg ToMV RNA in a 25-μl reaction mixture) and Western blot analysis to detect the 130K and 180K replication proteins. For RdRp product abbreviations, see the legend to Fig. 2A. Total RNA was also extracted from each fraction and subjected to agarose gel electrophoresis with ethidium bromide staining to detect rRNA or subjected to Northern hybridization to detect ToMV (−)RNA with a 32P-labeled P7P RNA probe (14).
FIG. 8.
FIG. 8.
Affinity purification of FLAG-tagged 180K replication protein. The iBYL S15 fraction (lanes 1, 2) or iBYL membranes (lanes 3, 4) from BY-2 cells were treated with LPC and subjected to affinity purification with anti-FLAG antibody-conjugated agarose beads, as described in Materials and Methods. In the BY-2 cells, replication of a ToMV derivative encoding either 180K-FLAG (lanes 2, 4), or nontagged 180K proteins (lanes 1, 3) was occurring. (A) Silver staining of affinity-purified proteins separated by SDS-12% PAGE. Equal volumes of purified samples were loaded in each lane. The positions and masses (in kDa) of protein markers are shown on the left. The expected locations of 180K-FLAG, 130 K, TOM1, TOM2A, eEF1A, and Hsp70 are shown on the right. (B) RdRp activity in the presence of exogenous ToMV RNA template. For RdRp product abbreviations, see the legend to Fig. 2A (the single-stranded genomic and subgenomic RNA bands were not detected here). (C) Detection of ToMV replication proteins, the TOM1 and TOM2A proteins, Hsp70, and eEF1A in the purified fractions. In panels B and C, the samples in lanes 1, 2, 3, and 4 were applied in a volume ratio of 1:1:4.3:4.3, respectively. This ratio yielded similar intensities of the 180K-FLAG bands on Western blots in lanes 2 and 4. The abbreviations are described in the legends to Fig. 2A and D for RdRp and Western analyses, respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bar-Peled, M., and N. V. Raikhel. 1997. Characterization of AtSEC12 and AtSAR1. Proteins likely involved in endoplasmic reticulum and Golgi transport. Plant Physiol. 114:315-324. - PMC - PubMed
    1. Blumenthal, T., and G. G. Carmichael. 1979. RNA replication: function and structure of Qβ replicase. Annu. Rev. Biochem. 48:525-548. - PubMed
    1. Browning, K. S., J. Humphreys, W. Hobbs, G. B. Smith, and J. M. Ravel. 1990. Determination of the amounts of the protein synthesis initiation and elongation factors in wheat germ. J. Biol. Chem. 265:17967-17973. - PubMed
    1. Buck, K. W. 1996. Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv. Vrius Res. 47:159-251. - PMC - PubMed
    1. Buck, K. W. 1999. Replication of tobacco mosaic virus RNA. Philos. Trans. R. Soc. Lond. B 354:613-627. - PMC - PubMed

Publication types