Complex dynamic development of poliovirus membranous replication complexes

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.

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

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

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

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

(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

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

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

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.