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. 2003 Aug;77(15):8512-23.
doi: 10.1128/jvi.77.15.8512-8523.2003.

Replication complex of human parechovirus 1

Camilla Krogerus  1 , Denise Egger, Olga Samuilova, Timo Hyypiä, Kurt Bienz
Affiliations

Replication complex of human parechovirus 1

Camilla Krogerus et al. J Virol. 2003 Aug.

Abstract

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

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Figures

FIG. 1.
FIG. 1.
Characterization of viral macromolecules in HPEV-1-infected cells. (A) Kinetics of viral RNA synthesis in infected cells determined by [3H]uridine labeling of nascent viral RNA. DPM, disintegrations per minute. (B) Immunoblots of mock-infected (lane 1) and HPEV-1-infected (lanes 2 to 4) cells (harvested at 4, 6, and 8 h p.i., respectively) were probed with Ab against protein 2C. Protein 2C accumulated in the absence of the precursor protein 2BC. (C) In vitro-translated proteins 2C (lane 1) and 2BC (lane 2) were immunoprecipitated with 2C antiserum and electrophoresed. The autoradiograph of the blot indicates that the 2C Ab was capable of recognizing 2C as well as the precursor protein 2BC.
FIG. 2.
FIG. 2.
Location of viral RNA and protein 2C in the infected cell. HPEV-1-infected A549 cells, harvested at 4 (a and e), 6 (b and f), 8 (c and g) and 10 (d and h) h p.i., were subjected to FISH with an FITC-coupled HPEV plus-strand specific riboprobe (a to d) or to IF with Ab against protein 2C (e to h). Bar, 25 μm.
FIG. 3.
FIG. 3.
Sequence comparison of human picornavirus 2C protein. (A) Phylogenetic tree of human picornaviruses based on the 2C genes. HRV, human rhinovirus; CAV, coxsackie A virus; CBV, coxsackie B virus. (B) Sequence alignment of the 2C proteins of HPEV-1, PV, and HAV. The three ATPase motifs are boxed, and 179N in the PV sequence and the corresponding glycine residues in the HPEV-1 and HAV sequences as well as 187M in the PV sequence and its corresponding amino acids in the HPEV-1 and HAV sequences, are highlighted. The cysteine-rich region in the PV sequence is underlined.
FIG. 4.
FIG. 4.
Electron micrographs of A549 cells demonstrate structural changes seen 6 h after HPEV-1 infection. (a) Uninfected cell; (b) infected cell, dashed line delineates region with alterations in the cellular architecture; (c and d) higher magnification shows the main features of cell alterations, including the appearance of clusters of small vesicles (V) (c), dilated ER (arrowheads), and rough ER (rER) (d). N, nucleus. Bars, 0.5 μm.
FIG. 5.
FIG. 5.
Location of protein 2C visualized by confocal scanning laser microscopy. (a) Double labeling of infected cell for protein 2C and Golgi protein GM130 shows the disintegration of the Golgi apparatus in the infected cell. Protein 2C was visualized with Texas Red-conjugated secondary Ab (red), and GM130 was visualized with Alexa 488-conjugated secondary Ab (green). 1, uninfected cell with intact Golgi; 2, HPEV-infected cell with dispersed Golgi complex. (b to f) Optical sectioning through an HPEV-1-infected cell 6 h p.i., stained with Ab against protein 2C. The distance between two consecutive sections is 0.5 μm. Protein 2C is located on branching, stick-like structures. (g to i) Double labeling of infected cells with Abs against protein 2C (red) and the cytoskeleton markers (green) tubulin (g) and vimentin (h) and with Alexa 488-coupled phalloidin that labels actin filaments (i). 1, uninfected cell with intact actin filaments; 2, HPEV-infected cell with depolymerized actin filaments. Picture sizes are as follows: a, 44 by 44 μm; b to f, 25 by 25 μm; g, 19 by 19 μm; h, 30 by 30 μm; i, 50 by 50 μm.
FIG. 6.
FIG. 6.
Localization of protein 2C in infected cells by IEM at 6 h p.i. (a) Overview of an infected cell showing longer and shorter narrow elongated labeled structures (arrowheads). At higher magnification, gold grains are found on stretches of membranes (arrowheads) (b) and on clusters of vesicles (arrowheads) (c). N, nucleus. Bars, 0.5 μm.
FIG. 7.
FIG. 7.
Membrane association of protein 2C determined by flotation assay and immunoblotting. (A) PNS of uninfected (I) and infected (II) cells 6 h p.i., before flotation. Lanes 1 to 12 (only even-numbered lanes are numbered), fractions of the sucrose gradient used for the flotation assay of untreated, infected cells. The membrane-binding proteins BAP31 and 2C are found on top of the gradient. (B) Flotation of Triton X-100-treated PNS. Proteins BAP31 and 2C remained at the bottom of the gradient. Lanes are labeled as for panel A.
FIG. 8.
FIG. 8.
Association of HPEV-1 RNA with small vesicles (arrowheads), shown by EM-ISH with a DIG-labeled riboprobe immunodetected with an anti-DIG Ab and 10-nm-diameter gold-conjugated secondary Ab. The inset shows an example of a cluster of vesicles. Bars, 0.2 μm.
FIG. 9.
FIG. 9.
Colocalization of nascent viral RNA with protein 2C or the trans-Golgi marker GalT in HPEV-1-infected, Br-UTP-transfected A549 cells at 6 h p.i. (a) Br-RNA (green) detected with an Ab against Br-dUTP and Alexa 488-conjugated secondary Ab. (b) Protein 2C (red) detected with an Ab against 2C and Texas Red-conjugated secondary Ab. (c) Merge of panels a and b. (d) Br-RNA (red) detected with an Ab against Br-dUTP and Texas Red-conjugated secondary Ab. (e) GalT (green) visualized with an anti-GalT Ab and FITC-conjugated secondary Ab. (f) Merge of panels d and e. Picture sizes, 25 by 25 μm.
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