Multiple pathways involved in porcine parvovirus cellular entry and trafficking toward the nucleus

Abstract

Porcine parvovirus (PPV) is a major cause of reproductive failure in swine. The mechanisms implicated in the first steps of infection that lead to the delivery of the PPV genome to the nucleus are poorly understood. In the present work, a panel of chemical inhibitors was used to dissect the cellular mechanisms involved in establishing a PPV infection. The results demonstrated that following binding to sialic acids on cell surface glycoproteins, the virus used both clathrin-mediated endocytosis and macropinocytosis pathways to gain access into cells. Virus obtained from infected cells was present either as isolated particles or as aggregates, and these two forms could be separated by low-speed centrifugation. Isolated and purified particles strongly preferred entry by clathrin-mediated endocytosis, whereas aggregates clearly favored macropinocytosis. Subsequent endosomal acidification and traffic to the late endosomes were also shown to be essential for infection. The microtubule network was found to be important during the first 10 h of infection, whereas an intact actin network was required for almost the whole viral cycle. Proteasome processing was found to be essential, and capsid proteins were ubiquitinated relatively early during infection. Taken together, these results provided new insights into the first steps of PPV infection, including the use of alternative entry pathways, unique among members of this viral family.

FIG. 1.

Kinetics of PPV infection. (A) Detection of viral capsids at different times p.i. in PT cells by immunofluorescence using the capsid-specific antibody 3C9 and an Alexa Fluor 488-coupled secondary antibody. (B) Amplification of the viral genome. qPCR specific to PPV was performed on cell lysates collected at different times p.i. (C) Cell DNA content in the presence of inhibitors, as an indication of side effects of the inhibitors on cell growth, determined by qPCR with c-myc gene-specific primers after 20 h of incubation with the optimal dose of each inhibitor.

FIG. 2.

PPV binding on the cell surface. (A) PT cells were treated with increasing amounts of neuraminidase in order to remove sialic acid moieties on cell surface glycoproteins. After a wash, PPV was added to the cells for 2 h. Unbound virus was removed by a wash at 2 h p.i., and infection was continued for an additional 18 h. The percentage of infected cells was compared to that of untreated cells (arbitrarily set at 100%) by IF with the capsid-specific antibody 3C9 and DNA staining (Hoechst). (B) Efficiency of infection recovery after neuraminidase treatment followed by specific reconstruction of sialic acids, using O- or N-sialyltransferases, on the cell surface proteins prior to infection. Percentages of infected cells were determined by IF (**, P < 0.004 by the t test).

FIG. 3.

Entry pathways. (A) Inhibition of the major cellular entry pathways during PPV infection. Optimal concentrations of inhibitors of clathrin endocytosis (chlorpromazine and K⁺ depletion) (light shaded bars), caveolae (nystatin and methyl-β-cyclodextrin) (filled bars), and macropinocytosis (amiloride and cytochalasin D) (dark shaded bars) were used. The percentage of cells infected was determined at 20 h by indirect IF experiments as described for Fig. 2. The combination of clathrin and macropinocytosis inhibitors (striped bars) failed to completely abolish infection (**, P < 0.003 by the t test). (B) Inhibition of genome replication measured by qPCR with specific PPV primers after treatment with inhibitors as for panel A (*, P < 0.03 by the t test). (C) Binding assay. Inhibitors were present only for the first 2 h with PPV at 4°C and were removed at the time of washing to eliminate unbound virus. The infection was continued in normal cell culture medium for 20 h. (D) Pulse inhibition. Clathrin endocytosis and macropinocytosis inhibitors either were added prior to infection and were present only for the first 2 h of infection, after which cells were washed; were added 2 h p.i.; or were added prior to infection and left during the whole infection (20 h). (E) Inhibition of distinct particle types. Shown are results for crude preparations (infected cell culture supernatants), isolated particles and aggregates (both separated by low-speed centrifugation of the crude preparation), and purified particles. To determine the inhibition of the clathrin and macropinocytosis pathways for each particle type, infections were carried out with the same amount of PPV, as determined by qPCR (***, P < 0.0003 by the t test).

FIG. 4.

Cytoplasm trafficking toward the nucleus. (A) Inhibitors of the endosomal pathway and acidification (light shaded bars), microtubules (filled bars), and actin networks (dark shaded bars) were used to evaluate their necessity for PPV infection. Shown are the relative percentages of infection at 20 h p.i. in the presence of optimal concentrations of the inhibitors as measured by indirect IF, as described for Fig. 2. (B) Impacts of the inhibitors on genome replication as measured by qPCR and normalized to cell numbers, as described for Fig. 3 (**, P < 0.003 by the t test). (C) Pulse inhibition. Inhibitors were added at different times p.i. and were left until 20 h p.i. The percentages of infected cells were determined (*, P < 0.03 by the t test for the difference between the Noc and LatA curves).

FIG. 5.

Proteasome involvement. (A and B) Inhibition of the proteolytic activity of the proteasome aborted PPV infection. The effects of the inhibitors were evaluated by indirect IF experiments as described for Fig. 2 and were expressed as the percentage of infected cells at 20 h p.i. relative to that for mock-treated cells (arbitrarily set at 100%). (C) Cells were treated with inhibitors as for panels A and B, and inhibition of genome replication was measured by qPCR for PPV, normalized to cell numbers, as described for Fig. 3. (D) Pulse inhibition. MG-132 was added at different times throughout the infection until cell fixation, and the percentage of infected cells was determined. (E) Capsid localization in the presence of MG-132. Capsids were visualized at different times throughout the infection by the capsid-specific antibody 3C9. In untreated cells, PPV reached perinuclear localization within 4 h p.i., and new virus was observed in the nucleus at 20 h p.i. The virus was also observed near the nucleus after MG-132-mediated inhibition but remained more diffuse in the cytoplasm (8 to 12 h p.i.), and very few positive nuclei were visible at 20 h p.i. (F) Coimmunoprecipitation of ubiquitin with anti-PPV capsid antibodies and of VP2 capsid proteins with anti-ubiquitin antibodies. Capsid proteins were observed in all ubiquitin precipitations. The ubiquitin signal in the capsid immunoprecipitation was more diffuse, as generally observed for ubiquitinated proteins.