Article

Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function.

Institute for Molecular Virology, University of Wisconsin, Madison, WI 53706, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2010; 107(37):16291-6. DOI: 10.1073/pnas.1011105107
Source: PubMed

ABSTRACT Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA replication is inhibited 80-90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2a(pol) polymerase. However, 1a failed to induce ER replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional replication vesicles but reduced their diameter by 30-50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2a(pol). Thus, RHPs interact with 1a, are incorporated into RNA replication compartments, and are required for multiple 1a functions in replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of replication vesicles.

Full-text

Available from: Arturo Diaz, Jun 16, 2015
0 Followers
 · 
112 Views
  • Future Virology 12/2014; 9(12):1089-1104. DOI:10.2217/fvl.14.95 · 1.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Replication of (+)RNA viruses depends on several co-opted host proteins, but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain containing proteins, which act as CIRFs. By using fusion proteins between the WW-domain and the p33 replication protein, we show that the WW-domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell-free extract. Over-expression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous co-expression of pro-viral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. In this paper, the authors define the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW-domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW-domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW-domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW-domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play pro-viral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus and it possibly has regulatory function during viral replication in infected cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Journal of Virology 12/2014; 89(4). DOI:10.1128/JVI.02719-14 · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.
    Virology 03/2015; 479-480. DOI:10.1016/j.virol.2015.02.029 · 3.28 Impact Factor