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
. 2012 Jan;86(1):302-12.
doi: 10.1128/JVI.05937-11. Epub 2011 Nov 9.

Complex dynamic development of poliovirus membranous replication complexes

George A Belov  1 , Vinod Nair, Bryan T Hansen, Forrest H Hoyt, Elizabeth R Fischer, Ellie Ehrenfeld
Affiliations

Complex dynamic development of poliovirus membranous replication complexes

George A Belov et al. J Virol. 2012 Jan.

Abstract

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as "vesicles" are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Virus-induced membrane remodeling in poliovirus-infected HeLa cells at various times postinfection. (A) Low-magnification transmission electron micrograph of a HeLa cell infected with poliovirus at 4 h.p.i. Inset: qPCR data showing kinetics of accumulation of viral RNA. (B, C, D, E, and F) Membranous structures in infected cells at 3, 4, 5, 6, and 7 h.p.i., respectively. Arrow, first detection of double-membrane structures in the middle of the infectious cycle (4 h.p.i.). Arrowhead, early single-membrane structures found at the late stage of infection (7 h.p.i.). (G) Cryo-SEM image showing clusters of early “saggy” membranous structures. Bars, 100 nm (unless otherwise indicated).
Fig 2
Fig 2
Poliovirus replication structures and cellular organelles. Images were taken at 3 h.p.i. (A) Viral replication structures are strongly associated with staining for a Golgi antigen, GM130. (B) Staining for a resident ER protein calnexin (arrowheads) that is excluded from virus replication membranes (arrows). Bar, 500 nm.
Fig 3
Fig 3
Golgi antigen GM130 is redistributed into multiple dot-like structures in poliovirus-infected cells. HeLa cells were infected for 6 h with a poliovirus mutant with an HA tag within the 3A protein. Poliovirus-infected (A to C) and mock-infected (D to F) cells were stained simultaneously with anti-HA and anti-GM130 antibodies.
Fig 4
Fig 4
Reconstructions showing the 3-D architecture of poliovirus membranous replication complexes at the early (3 h.p.i.), intermediate (4 h.p.i.), and late (7 h.p.i.) stages of development. (A, D, and G) Central slices in tomographic volumes. (B, E, and H) Central slices with segmented overlays. (C, F, and I) Segmented volumes, with blue indicating single-membrane structures and yellow and green indicating inner and outer membranes of double-membrane structures, respectively. Bar, 100 nm.
Fig 5
Fig 5
(A) A cryo-SEM image of freeze-fractured HeLa cells infected with poliovirus, showing late spherical structures at 6 h.p.i. (B) A cryo-SEM image of HeLa cells infected with poliovirus osmicated before freeze fracturing, revealing the inner contents of late spherical membrane structures. (C) Higher magnification of the region of interest demarcated in panel B. Bars, 1 μm (A and B) and 100 nm (C).
Fig 6
Fig 6
Late double-membrane structures form by wrapping of early single-membrane membranous chambers. Slices (1.4 nm) through tomographic reconstructions are shown. Bead masking during tilt series alignment (IMOD) and 3-D noise-reduction median filtering (Amira) were applied for visualization. (A, B, and C) Slices from a single volume, showing coexistence of open single-membrane configurations and closed double-membrane regions in continuous membranous structures in infected cells. Cells were fixed at 4 h.p.i. (D, E, and F) Slices from a second volume, showing a potential region of closure that appears to have engulfed cytoplasmic granules. Cells were fixed at 6 h.p.i. Bar, 100 nm.
Fig 7
Fig 7
Poliovirus replication proteins and dsRNAs are associated with both single- and double-membrane structures. (A, B, and C) HeLa cells infected with poliovirus at 4 h.p.i. were stained with antibodies against poliovirus protein 3A (A) or 2C (B) or dsRNA (C). Arrows show single-membrane structures; arrowheads show double-membrane structures. (D) Background staining of infected cells processed without primary antibody. Bar, 100 nm.
Fig 8
Fig 8
Actively replicating poliovirus RNA is associated with single- and double-membrane structures. (A) HeLa cells infected with poliovirus were pulsed-labeled with BrUTP from 3.5 to 4.5 h.p.i. and stained with anti-BrdUTP antibodies. Arrows point to labeled single-membrane structures; arrowheads show labeled double-membranes structures. ER, endoplasmic reticulum; MT, mitochondrion; N, nucleus. (B) Background staining control. Infected cells processed without primary antibody are shown. (C) qPCR data reflecting kinetics of accumulation of viral RNA. Bar, 100 nm.
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, et al. 1985. Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses. J. Virol. 53:536–542 - PMC - PubMed
    1. Agirre A, Barco A, Carrasco L, Nieva JL. 2002. Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein. J. Biol. Chem. 277:40434–40441 - PubMed
    1. Amako K, Dales S. 1967. Cytopathology of mengovirus infection. II. Proliferation of membranous cisternae. Virology 32:201–215 - PubMed
    1. Baumeister W, Grimm R, Walz J. 1999. Electron tomography of molecules and cells. Trends Cell Biol. 9:81–85 - PubMed
    1. Belov GA, et al. 2007. Hijacking components of the cellular secretory pathway for replication of poliovirus RNA. J. Virol. 81:558–567 - PMC - PubMed

Publication types