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. 2001 Feb;75(4):1958-67.
doi: 10.1128/JVI.75.4.1958-1967.2001.

Entry of human parechovirus 1

P Joki-Korpela  1 , V Marjomäki, C Krogerus, J Heino, T Hyypiä
Affiliations

Entry of human parechovirus 1

P Joki-Korpela et al. J Virol. 2001 Feb.

Abstract

Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes alpha(V) integrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against alpha(V) and beta(3) integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPEV-1 infection. To find out whether HPEV-1 uses clathrin-coated vesicles or other routes for the entry into the host cell, we carried out double-labeling experiments of virus-infected cells with anti-HPEV-1 antibodies and antibodies against known markers of the clathrin and the caveolin routes. At the early phase of infection (5 min postinfection [p.i.]) HPEV-1 colocalized with EEA1 (early endosomes), and later, after 30 min p.i., it colocalized with mannose-6-phosphate receptor (late endosomes), whereas no colocalization with caveolin-1 was observed. The data indicate that HPEV-1 utilizes the clathrin-dependent endocytic pathway for entry into the host cells. Interestingly, endocytosed HPEV-1 capsid proteins were observed in the endoplasmic reticulum and cis-Golgi network 30 to 60 min p.i. Depolymerization of microtubules with nocodazole inhibited translocation of the virus to the late endosomes but did not block HPEV-1 replication, suggesting that the RNA genome may be released early during the entry process.

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Figures

FIG. 1
FIG. 1
Production of infectious virus during HPEV-1 infection in A549 cells. The amount of virus was determined from the cells and the culture supernatants using plaque assay.
FIG. 2
FIG. 2
Localization of HPEV-1 capsid proteins in A549 cells during the infection cycle, visualized by indirect immunofluorescence. Rabbit antiserum against purified HPEV-1 was used as the primary antibody. Bars, 10 μm.
FIG. 3
FIG. 3
Blocking of HPEV-1 infection by Abs against different cell surface proteins. αV, α2, β1, β2 microglobulin (β2-m), and DAF Abs were used at a dilution of 1:100 while anti-CAR and -β3 supernatants were used undiluted. After incubation with the Abs, the cells were infected with the virus (100 PFU), and the number of plaques was counted after 48 h of incubation. The results are expressed as proportional numbers of plaques compared to those appearing in the cells infected in the absence of Abs.
FIG. 4
FIG. 4
Localization of HPEV-1 capsid proteins and integrin subunits (α2, αV, β1, and β3) during the early steps of infection. Red, virus; green, integrin subunits; yellow, colocalization. Bars, 10 μm.
FIG. 5
FIG. 5
Colocalization of HPEV-1 with early and late endocytic markers. EEA1 was used to stain the early endosomes at 5 and 15 min p.i. CI-MPR was used for the detection of late endosomes at 30 min p.i. in A549 cells with or without nocodazole (noc) treatment. Green, virus; red, EEA1 or CI-MPR; yellow, colocalization. Bars, 10 μm.
FIG. 6
FIG. 6
Subcellular fractionation of cellular homogenates from HPEV-1-infected cells in 20% Percoll gradients. A549 cells were allowed to internalize HRP for 5 min at 37°C in order to label early endosomes. Three adjoining fractions were pooled from the bottom (fractions 5 to 7; pool I), from the middle (fractions 10 to 12; pool II), and from the top (fractions 18 to 20; pool III) of the gradient. The pooled fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using HPEV-1 rabbit Abs.
FIG. 7
FIG. 7
Colocalization of HPEV-1 capsid proteins with ER (1D3 Ab) at 30 and 60 min p.i. 1D3 detects protein disulfide isomerase in the endoplasmic reticulum. C, uninfected control. Red, virus; green, 1D3; yellow, colocalization. Bars, 10 μm.
FIG. 8
FIG. 8
Colocalization of HPEV-1 capsid proteins with cis-Golgi network (p23 Ab) at 30 and 60 min p.i. C, uninfected control. Green, virus; red, p23; yellow, colocalization. Bars, 10 μm.
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