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. 2003 Jan;77(1):460-9.
doi: 10.1128/jvi.77.1.460-469.2003.

Role of protein kinase C betaII in influenza virus entry via late endosomes

Sara B Sieczkarski  1 , H Alex Brown, Gary R Whittaker
Affiliations

Role of protein kinase C betaII in influenza virus entry via late endosomes

Sara B Sieczkarski et al. J Virol. 2003 Jan.

Abstract

Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C betaII (PKCbetaII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCalpha and -beta. Cells expressing a phosphorylation-deficient form of PKCbetaII (T500V), but not an equivalent mutant form of PKCalpha, inhibited successful influenza virus entry-with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCbetaII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCbetaII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCbetaII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCbetaII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.

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Figures

FIG. 1.
FIG. 1.
PKC inhibitor Gö6976 prevents influenza virus entry. Immunofluorescence assay of inhibitor treated cells. HeLa cells were infected for 4 h at a low multiplicity of infection in the presence of 20 μM Gö6976 (a) or left untreated (b). Influenza infection was detected by the monoclonal NP antibody. Bars = 5 μm.
FIG. 2.
FIG. 2.
Expression of phosphorylation-deficient PKCβII, but not PKCα, prevents influenza virus entry. (A) HeLa cells were transiently transfected with wild-type and phosphorylation-deficient T500V PKCβII before infection at a high multiplicity of infection for 60 min. (B) Mv1 Lu, CHO, BHK, and polarized MDCK cells were transiently transfected with PKCβII T500V plasmid before a high-multiplicity infection for 60 min. (C) HeLa cells were transiently transfected with wild-type or phosphorylation-deficient (K368R) PKCα plasmids before infection at a high multiplicity of infection for 60 min. Control cells were not transfected before infection. Influenza virus signal was detected via indirect immunofluorescence using a polyclonal NP antibody. Expression was detected using a monoclonal antibody against PKCβ or FLAG epitope. Bars = 5 μm.
FIG. 3.
FIG. 3.
SFV infection is not inhibited by expression of PKCβII T500V. HeLa cells were transiently transfected with the PKCβII wild-type or T500V plasmid for 16 h before infection. Cells were infected with SFV (a and b) or influenza virus (c and d) at a low multiplicity of infection for 5 h. Virus signal was detected by the monoclonal anti-SFV E1-1 antibody or anti-influenza virus NP antibody. PKCβII expression was detected by a monoclonal antibody against PKCβ. Bars = 5 μm.
FIG. 4.
FIG. 4.
Influenza vRNPs accumulate in late endosomes in cells lacking PKCβII activity. HeLa cells were transiently transfected with the PKCβII wild-type or T500V plasmids for 16 h before a high-multiplicity infection for 60 min. (A) Indirect immunofluorescence studies were used to analyze the expression pattern of influenza virus and cellular vesicles. Influenza virus localization was determined using a polyclonal NP antibody. EEA1 (a marker for early endosomes), M6PR and CD63/Lamp3 (markers for late endosomes), and the Golgi apparatus were localized using their respective monoclonal antibodies. (B) Confocal microscopy studies were used to analyze the expression pattern of influenza virus and late endocytic vesicles. Influenza localization was determined by the polyclonal NP antibody, and late endosomes were localized with a monoclonal antibody to CD63/Lamp3. Bars = 5 μm.
FIG. 5.
FIG. 5.
Expression of PKCβII T500V does not affect endosome acidification needed for virus entry. (A) HeLa cells were transiently transfected with the PKCβII wild-type or T500V plasmid for 16 h before a high-multiplicity infection for 60 min. Cells were then treated with 10 μM nigericin and monensin and incubated in medium buffered to either pH 7.0 or 5.5 with 20 mM HEPES. Cells were fixed at 60 min, and vRNPs were detected via indirect immunofluorescence using a polyclonal NP antibody. Bars = 5 μm. (B) HeLa cells were transiently transfected with the PKCβII wild-type or T500V plasmid for 16 h before a high-multiplicity infection with the X-31 strain of influenza virus. The viral HA glycoprotein was detected by indirect immunofluorescence with the conformation-specific monoclonal antibodies N2 (neutral) and A2 (acidic). Controls were not transfected but were treated with 25 nM bafilomycin A. Bars = 8 μm.
FIG. 6.
FIG. 6.
EGFR is less efficiently degraded in cells expressing PKCβII T500V. (A) HeLa cells were transiently transfected with the wild-type or kinase-dead (T500V) PKCβII plasmid for 16 h. Cells were then serum starved for 24 h at 37°C. EGF was bound at 4°C for 60 min, and cells were washed and then incubated at 37°C for the indicated times. EGFR was detected by indirect immunofluorescence using an anti-EGFR monoclonal antibody. Bars = 8 μm. (B) HeLa cells were transiently transfected with the PKCβII T500V plasmid for 16 h or were mock transfected. Cells were serum starved, and the EGF uptake assay was performed at 1 h (blue trace) and 3 h (red trace). Cells were prepared for flow cytometry using the monoclonal antibody against the EGFR. (C) HeLa cells were transiently transfected with the kinase-dead (T500V) PKCβII, kinase-dead PKCα (K368R), or dominant-negative PKCλ (λ/ι PKCMUT) plasmid for 16 h or were untransfected. Cells were serum starved, and the EGF uptake assay was performed as described for panel B. Cells were prepared for flow cytometry using the monoclonal antibody against the EGFR. The signal intensity was normalized at the 1 h time point, and the relative amount of EGFR degraded by the 3 h time point is shown as a percentage. (D) HeLa cells were transiently transfected with the PKCβII T500V plasmid for 16 h and then serum starved. EGF uptake was allowed to occur for 120 min. Confocal microscopy studies were used to analyze the expression pattern of the EGFR and late endocytic vesicles. EGFR localization was determined using an anti-EGFR antibody, and late endosomes were localized with an antibody to CD63/Lamp3. Bars = 5 μm.
FIG. 7.
FIG. 7.
Transferrin receptor recycling is unaffected by the lack of PKCβII activity. HeLa cells were transiently transfected with the PKCβII wild-type or T500V plasmid for 16 h. Alexa 594-labeled transferrin (50 μg/ml) was then bound to serum-starved cells for 20 min at 4°C. Cells were then shifted to 37°C for 15 min, washed in low-pH glycine to remove any uninternalized ligand, and returned to 37°C for 15 min to monitor receptor recycling. Cells were analyzed by fluorescence microscopy with an antibody to transferrin receptor. Bars = 8 μm.
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