Identification of a role for the trans-Golgi network in human papillomavirus 16 pseudovirus infection

Abstract

Human papillomavirus 16 (HPV16) enters its host cells by a process that most closely resembles macropinocytosis. Uncoating occurs during passage through the endosomal compartment, and the low pH encountered in this environment is essential for infection. Furin cleavage of the minor capsid protein, L2, and cyclophilin B-mediated separation of L2 and the viral genome from the major capsid protein, L1, are necessary for escape from the late endosome (LE). Following this exodus, L2 and the genome are found colocalized at the ND10 nuclear subdomain, which is essential for efficient pseudogenome expression. However, the route by which L2 and the genome traverse the intervening cytoplasm between these two subcellular compartments has not been determined. This study extends our understanding of this phase in PV entry in demonstrating the involvement of the Golgi complex. With confocal microscopic analyses involving 5-ethynyl-2'-deoxyuridine (EdU)-labeled pseudogenomes and antibodies to virion and cellular proteins, we found that the viral pseudogenome and L2 travel to the trans-Golgi network (TGN) following exit from the LE, while L1 is retained. This transit is dependent upon furin cleavage of L2 and can be prevented pharmacologically with either brefeldin A or golgicide A, inhibitors of anterograde and retrograde Golgi trafficking. Additionally, Rab9a and Rab7b were determined to be mediators of this transit, as expression of dominant negative versions of these proteins, but not Rab7a, significantly inhibited HPV16 pseudovirus infection.

Fig 1

Time course of L1 and pseudogenome colocalization. HeLa cells were incubated with HPV16 pseudovirus that contained EdU-labeled pseudogenome. Cells were fixed after various incubation times, as indicated, following pseudovirus addition. The antigens detected are also indicated within the individual panels. The merged images are in the rightmost panel of each row. The nuclei were detected with DAPI (blue) in all panels.

Fig 2

Localization of pseudogenome relative to subcellular markers. Cells were harvested at 24 h post-virus addition. Reagents used for detection are indicated within the individual panels. Please note that panel A shows HPV capsid staining, whereas all other panels show EdU detection, relative to subcellular markers. The nuclei were detected with DAPI (blue) in all panels.

Fig 3

Inhibition profiles of BFA and GCA. HaCaT cells were incubated with a dilution series of either BFA or GCA, and infection with a GFP-expressing pseudovirus was allowed to continue for 48 h. Following this, GFP expression was determined by flow cytometric analysis. The percent inhibition was determined by comparison to control infections. The results from triplicate infections are shown. The starting concentration of BFA was 150 nM, and 11 3-fold dilutions were examined. The starting concentration of GCA was 5 μM, and 11 2-fold dilutions were examined.

Fig 4

Effects of BFA and GCA on pseudogenome trafficking. The localization of EdU-labeled pseudogenome was analyzed relative to the Lamp1 protein. Cells were left untreated (untx) or incubated in the presence of either BFA or GCA, as indicated. Merged images are shown in the rightmost panel of each row. Cells were harvested at 24 h post-virus addition. The nuclei were detected with DAPI (blue) in all panels.

Fig 5

Dependence on furin-cleaved L2 for TGN localization. HA-tagged L2 was localized relative to the EdU-labeled genome (A) and gm130 (B) as indicated. EdU detection from L1-only VLPs is shown in panels C and D relative to p230 and L1 as indicated. EdU detection from pseudovirus containing an L2 furin cleavage mutant is shown in panels E and F relative to L1 and p230 as indicated. Cells were harvested at 24 h postentry. The nuclei were detected with DAPI (blue) in all panels.

Fig 6

Effect of expression of DN Rab proteins on HPV16 pseudovirus infection. (A) 293TT cells were transfected with WT or DN Rab expression plasmids. At 24 h, these cells were infected with HPV16 pseudovirus containing a GFP expression plasmid (for Rab7a and Rab9a) or an RFP expression plasmid (for Rab7b). Following 48 h of infection, RFP and GFP coexpression was determined. The percentage of transfected cells that were also infected was determined. The plain bar indicates this percentage for the WT protein; the bar with the horizontal stripes represents the DN protein. Rab 7a is shown in white, Rab 9a in light gray, and Rab 7b in dark gray. (B) The transfected cells were either incubated with exogenous furin during the infection (left side of graph) or plated over furin-cleaved pseudovirus in the presence of furin inhibitor (right side of graph).