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Clathrin- and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs).

Spoden G, Freitag K, Husmann M, Boller K, Sapp M, Lambert C, Florin L - PLoS ONE (2008)

Bottom Line: Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection.However, their involvement in endocytosis of viral particles has not been proven.Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University of Mainz, Mainz, Germany.

ABSTRACT

Background: Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.

Methodology/principal findings: Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.

Conclusions/significance: Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

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Dynamin-independent entry and infection of HPV16 PsVs.(A) HeLa cells were either mock treated (control), treated with 80 µM dynasore for 30 min, transfected with dynamin2 specific siRNA for 48 hours (Dynamin siRNA), or transfected with GFP-tagged dynamin2 K44A mutant for 24 hours (Dyn2 K44A-GFP). Thereafter, cells were incubated with AlexaFluor546 labeled transferrin or infected with HPV16 pseudovirions for 6 hours (dynasore: 4 hours). Cells were fixed and permeabilized with methanol. Immunostaining was performed with the indicated L1-specific antibody (L1-7, red). Cells were examined by immunofluorescence microscopy. Bar, 20 µm. (B) siRNA mediated knockdown of dynamin2 (Dyn2) in HeLa and 293TT cells was controlled 48 hours after transfection by Western blotting. (C, D) Infection assay was performed in dynamin2 or control siRNA transfected as well as in dn dynamin2 mutant (Dyn2 K44A) or control transfected 293TT (dark gray) and HeLa (light gray) cells. Infectivity was quantified by FACS (n = 4, +/−SD); infection rate of the control was set to 100%.
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pone-0003313-g004: Dynamin-independent entry and infection of HPV16 PsVs.(A) HeLa cells were either mock treated (control), treated with 80 µM dynasore for 30 min, transfected with dynamin2 specific siRNA for 48 hours (Dynamin siRNA), or transfected with GFP-tagged dynamin2 K44A mutant for 24 hours (Dyn2 K44A-GFP). Thereafter, cells were incubated with AlexaFluor546 labeled transferrin or infected with HPV16 pseudovirions for 6 hours (dynasore: 4 hours). Cells were fixed and permeabilized with methanol. Immunostaining was performed with the indicated L1-specific antibody (L1-7, red). Cells were examined by immunofluorescence microscopy. Bar, 20 µm. (B) siRNA mediated knockdown of dynamin2 (Dyn2) in HeLa and 293TT cells was controlled 48 hours after transfection by Western blotting. (C, D) Infection assay was performed in dynamin2 or control siRNA transfected as well as in dn dynamin2 mutant (Dyn2 K44A) or control transfected 293TT (dark gray) and HeLa (light gray) cells. Infectivity was quantified by FACS (n = 4, +/−SD); infection rate of the control was set to 100%.

Mentions: As it has been shown that the internalization of clathrin-coated vesicles and the uptake of caveolar microdomains are dependent on the functionality of the large GTPase dynamin we additionally analyzed the impact of dynamin for HPV16 entry and infection. In a first approach we inhibited dynamin function by treatment of HeLa cells with the inhibitor dynasore [34]. Whereas uptake of transferrin was efficiently inhibited (Figure 4A, Dynasore, upper panel), we found effective entry of HPV16 PsVs as indicated by the reactivity of the monoclonal antibody L1-7 (Figure 4A, Dynasore, lower panel). The same results were obtained using depletion of dynamin-2 by specific siRNA treatment (Figure 4A, Dynamin siRNA). The effectiveness of dynamin-2 depletion in HeLa cells was controlled by Western blot analysis (Figure 4B). In a third approach we specifically blocked the functionality of dynamin-2 by expression of the GPF-tagged dominant negative GTPase-deficient mutant Dyn2K44A-GFP [35]–[37]. Again, endocytosis of transferrin was inhibited in cells expressing the dominant-negative mutant, whereas transferrin was efficiently taken up in non-transfected cells (Figure 4A, Dyn2K44A-GFP, upper panel). However, entry of virions was not affected by expression of Dyn2K44A (Figure 4B, Dyn2K44A-GFP, lower panel). In order to test whether this dynamin-independent entry of virions results in productive infection, we performed infection assays in 293TT and HeLa cells. As shown in figure 4C, we found no inhibitory effect on infection efficiency when dynamin-2 was depleted with siRNA, or when we expressed the dn Dyn2K44A-mutant (Figure 4D). Rather, in any case we repeatedly observed a slight increase in the number of infected cells. These data confirmed our observations that entry and infection of HPV16 occur independently of clathrin- and caveolin-mediated endocytosis, since both pathways require the function of the large GTPase dynamin.


Clathrin- and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs).

Spoden G, Freitag K, Husmann M, Boller K, Sapp M, Lambert C, Florin L - PLoS ONE (2008)

Dynamin-independent entry and infection of HPV16 PsVs.(A) HeLa cells were either mock treated (control), treated with 80 µM dynasore for 30 min, transfected with dynamin2 specific siRNA for 48 hours (Dynamin siRNA), or transfected with GFP-tagged dynamin2 K44A mutant for 24 hours (Dyn2 K44A-GFP). Thereafter, cells were incubated with AlexaFluor546 labeled transferrin or infected with HPV16 pseudovirions for 6 hours (dynasore: 4 hours). Cells were fixed and permeabilized with methanol. Immunostaining was performed with the indicated L1-specific antibody (L1-7, red). Cells were examined by immunofluorescence microscopy. Bar, 20 µm. (B) siRNA mediated knockdown of dynamin2 (Dyn2) in HeLa and 293TT cells was controlled 48 hours after transfection by Western blotting. (C, D) Infection assay was performed in dynamin2 or control siRNA transfected as well as in dn dynamin2 mutant (Dyn2 K44A) or control transfected 293TT (dark gray) and HeLa (light gray) cells. Infectivity was quantified by FACS (n = 4, +/−SD); infection rate of the control was set to 100%.
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Related In: Results  -  Collection

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pone-0003313-g004: Dynamin-independent entry and infection of HPV16 PsVs.(A) HeLa cells were either mock treated (control), treated with 80 µM dynasore for 30 min, transfected with dynamin2 specific siRNA for 48 hours (Dynamin siRNA), or transfected with GFP-tagged dynamin2 K44A mutant for 24 hours (Dyn2 K44A-GFP). Thereafter, cells were incubated with AlexaFluor546 labeled transferrin or infected with HPV16 pseudovirions for 6 hours (dynasore: 4 hours). Cells were fixed and permeabilized with methanol. Immunostaining was performed with the indicated L1-specific antibody (L1-7, red). Cells were examined by immunofluorescence microscopy. Bar, 20 µm. (B) siRNA mediated knockdown of dynamin2 (Dyn2) in HeLa and 293TT cells was controlled 48 hours after transfection by Western blotting. (C, D) Infection assay was performed in dynamin2 or control siRNA transfected as well as in dn dynamin2 mutant (Dyn2 K44A) or control transfected 293TT (dark gray) and HeLa (light gray) cells. Infectivity was quantified by FACS (n = 4, +/−SD); infection rate of the control was set to 100%.
Mentions: As it has been shown that the internalization of clathrin-coated vesicles and the uptake of caveolar microdomains are dependent on the functionality of the large GTPase dynamin we additionally analyzed the impact of dynamin for HPV16 entry and infection. In a first approach we inhibited dynamin function by treatment of HeLa cells with the inhibitor dynasore [34]. Whereas uptake of transferrin was efficiently inhibited (Figure 4A, Dynasore, upper panel), we found effective entry of HPV16 PsVs as indicated by the reactivity of the monoclonal antibody L1-7 (Figure 4A, Dynasore, lower panel). The same results were obtained using depletion of dynamin-2 by specific siRNA treatment (Figure 4A, Dynamin siRNA). The effectiveness of dynamin-2 depletion in HeLa cells was controlled by Western blot analysis (Figure 4B). In a third approach we specifically blocked the functionality of dynamin-2 by expression of the GPF-tagged dominant negative GTPase-deficient mutant Dyn2K44A-GFP [35]–[37]. Again, endocytosis of transferrin was inhibited in cells expressing the dominant-negative mutant, whereas transferrin was efficiently taken up in non-transfected cells (Figure 4A, Dyn2K44A-GFP, upper panel). However, entry of virions was not affected by expression of Dyn2K44A (Figure 4B, Dyn2K44A-GFP, lower panel). In order to test whether this dynamin-independent entry of virions results in productive infection, we performed infection assays in 293TT and HeLa cells. As shown in figure 4C, we found no inhibitory effect on infection efficiency when dynamin-2 was depleted with siRNA, or when we expressed the dn Dyn2K44A-mutant (Figure 4D). Rather, in any case we repeatedly observed a slight increase in the number of infected cells. These data confirmed our observations that entry and infection of HPV16 occur independently of clathrin- and caveolin-mediated endocytosis, since both pathways require the function of the large GTPase dynamin.

Bottom Line: Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection.However, their involvement in endocytosis of viral particles has not been proven.Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University of Mainz, Mainz, Germany.

ABSTRACT

Background: Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.

Methodology/principal findings: Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.

Conclusions/significance: Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

Show MeSH
Related in: MedlinePlus