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. 2018 Dec 26;13(12):e0208743.
doi: 10.1371/journal.pone.0208743. eCollection 2018.

Cowpea chlorotic mottle bromovirus replication proteins support template-selective RNA replication in Saccharomyces cerevisiae

Bryan S Sibert  1   2 , Amanda K Navine  1   3 , Janice Pennington  1   2 , Xiaofeng Wang  1 , Paul Ahlquist  1   2   3
Affiliations

Cowpea chlorotic mottle bromovirus replication proteins support template-selective RNA replication in Saccharomyces cerevisiae

Bryan S Sibert et al. PLoS One. .

Abstract

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others' genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the replication complex. Overall, we show that many 1a and 2apol functions essential for replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the replication complex.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. DNA launched CCMV 1a, 2a, RNA3, and RNA2 expression and replication.
(A) The open reading frames for CCMV 1a and 2a from RNA1 and 2 respectively were cloned in between the yeast GAL1 promoter and the yeast ADH1 terminator. These constructs will express the viral proteins, but lack essential non-coding viral sequence required for RNA replication. Highly conserved regions of 1a responsible for methyltransferase activity (diamonds), membrane association (‘M’), and predicted helicase domains (vertical bars) are indicated. The core RNA-dependent RNA-polymerase domain of 2apol is indicated by striping. (B) Full CCMV RNA3 cDNA sequence was cloned between the GAL1 promoter and a ribozyme (Rz) derived from hepatitis delta virus that cleaves itself from the RNA leaving the natural viral 3’end. CCMV 1a and 2apol proteins direct negative-strand synthesis of RNA3, which then serves as a template for additional positive strand synthesis of both RNA3 and the subgenomic RNA4. Expression of the coat protein depends on synthesis of (+)RNA4 from the (-)RNA3 template, but is intentionally blocked in this construct by an introduced frameshift mutation. (C) Full-length CCMV RNA2 cDNA was cloned between the GAL1 promoter and ribozyme as for CCMV RNA3. A small deletion in this construct disrupts the 2apol ORF and blocks cis- expression of 2a from the template.
Fig 2
Fig 2. Virus and RNA template specific differences in RNA replication levels in yeast.
(A) Total RNA from yeast expressing BMV RNA3 alone, lanes 1–3; BMV RNA3, BMV 1a, and BMV 2a, lanes 3–6; or BMV RNA3, CCMV 1a, and CCMV 2a, lanes 7–9 was probed for positive and negative- strand RNA3 and 4 as indicated by northern blotting. Relative levels of RNA3 are indicated beneath the blots. Blots are shown to the right with strongly enhanced contrast to better visualize replication dependent bands in lanes 7–9. 18S RNA is shown as a loading control. Bands other than (+)RNA3 present in the absence of RNA replication are marked with a an asterisk. (B) As in A, except with a CCMV RNA3 template in place of BMV RNA3. The average and standard deviation of each triplicate shown is presented beneath the blots. A line connecting two numbers indicates a statistically significant difference (p<0.05).
Fig 3
Fig 3. CCMV 2a polymerase is competent for negative-strand RNA synthesis in yeast.
Northern blotting was used to detect viral RNA from yeast expressing BMV RNA3 with different combinations of BMV or CCMV 1a and 2apol as indicated. 18S RNA is shown as a loading control. Bands other than (+)RNA3 present in the absence of RNA replication are marked with a an asterisk. The average and standard deviation of each triplicate shown is presented beneath the blots. A line connecting two numbers indicates a statistically significant difference (p<0.05).
Fig 4
Fig 4. CCMV 1a is defective relative to BMV 1a in recruiting RNA templates to membranes.
(A) Positive-strand BMV or CCMV RNA3 accumulation was detected by northern blotting from cells expressing RNA3 alone or with BMV or CCMV 1a as indicated. (B,C) As in (A) except yeast were lysed and centrifuged to pellet cellular membranes prior to RNA isolation. Accumulation of positive-strand RNA3 was assayed from total lysate (T), supernatant (S), and pellet (P) fractions. The relative percentage of RNA in the pellet fraction is indicated below each gel as the average and standard deviation quantified from three fractionations. A line connecting two numbers indicates a statistically significant difference (p<0.05).
Fig 5
Fig 5. 1a-dependent RNA accumulation and replication of bromovirus RNA2 templates in yeast.
(A) Northern blot from total RNA from yeast expressing BMV RNA2 alone or with BMV 1a and/or 2a or CCMV 1a and/or 2a as indicated. Relative levels of RNA2 are indicated beneath the blots. (B) As in (A) except with a CCMV RNA2 template in place of BMV RNA3. The average and standard deviation of three replicates is shown beneath the lanes. A line connecting two numbers indicates a statistically significant difference (p<0.05).
Fig 6
Fig 6. CCMV 1a recruits RNA2 templates to membranes.
(A) Cells expressing BMV or CCMV RNA2 alone or with BMV or CCMV 1a as indicated were lysed and centrifuged to pellet cellular membranes prior to RNA isolation. Accumulation of positive-strand RNA2 was assayed by northern blotting from total lysate (T), supernatant (S), and pellet (P) fractions. The relative percentage of RNA in the pellet fraction is indicated below each gel as the average and standard deviation quantified from three fractionations. A line connecting two numbers indicates a statistically significant difference (p<0.05).
Fig 7
Fig 7. CCMV 1a localizes to the perinuclear ER and recruits CCMV 2a.
(A) Replication of BMV RNA3 by BMV and CCMV with untagged 2a and GFP-2a was detected by northern blotting. (B) Western blotting was performed on total protein lysate from yeast expressing BMV 1a and GFP-BMV 2a or CCMV 1a and GFP-CCMV 2a using the antibodies indicated. Pgk1p is an endogenous yeast protein used as a loading control. The average and standard deviation of three replicates is shown beneath the lanes. A line connecting two numbers indicates a statistically significant difference (p<0.05). (C) Fluorescence confocal microscopy was used to image cells expressing BMV 1a (red) and Sec63-GFP (ER marker), BMV GFP-2a (green) only, or BMV 1a and BMV GFP-2a. DNA was stained with DAPI as a nuclear marker. (D,E) As for (B) with (D) CCMV 1a and CCMV GFP-2a or (E) the heterologous 1a/2a combinations as indicated. All images are projections of a confocal z-stack.
Fig 8
Fig 8. CCMV 1a is sufficient to induce spherule membrane rearrangements along the ER.
Yeast expressing BMV 1a (A) or CCMV 1a (B) were prepared for EM using conventional chemical fixation and embedding (see Methods for details). Arrows identify 1a protein induced invaginations along the perinuclear ER. (C, D, E) Representative images of yeast expressing only the non-coding BMV RNA3 template (C) or BMV 1a (D) or CCMV 1a (E) prepared by high-pressure freezing and freeze substitution. All scale bars are 100 nm.
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P.A. is an investigator of the Howard Hughes Medical Institute and the Morgridge Institute for Research, and gratefully acknowledges support from these institutes, the NIH, and the John W. and Jeanne M. Rowe Virology Venture Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.