Noncanonical Rab9a Action Supports Retromer-mediated Endosomal Exit of Human Papillomavirus During Virus Entry

Jeongjoon Choi, Daniel DiMaio


Rab GTPases play key roles in controlling intracellular vesicular transport. GTP-bound Rab proteins support vesicle trafficking. Here, we report that, unlike cellular protein cargos, retromer-mediated delivery of human papillomaviruses (HPV) into the retrograde transport pathway during virus entry is inhibited by Rab9a in its GTP-bound form. Knockdown of Rab9a inhibits HPV entry by modulating the HPV-retromer interaction and impairing retromer-mediated endosome-to-Golgi transport of the incoming virus, resulting in the accumulation of HPV in the endosome.


Rab GTPases are key regulators of intracellular vesicular transport [1–3]. In general, when bound to guanosine triphosphate (GTP), Rab proteins are active and support vesicle trafficking, but when bound to guanosine diphosphate (GDP) they do not support vesicle transport [1,3]. Trafficking of some viruses to the site of viral genome replication depends on cellular Rab proteins [4–6]. Human papillomaviruses (HPVs) are non-enveloped, double-stranded DNA viruses that trigger ~5% of human cancer, including essentially all cervical cancer [7]. HPV entry requires various Rab proteins, but it remains largely unclear which Rab proteins are employed at which specific steps of virus entry [6]. Here, we show that GDP-bound Rab9a is necessary for HPV trafficking from the endosome to the trans-Golgi network (TGN) during entry.

Materials and method

HeLa S3 cells were purchased from American Type Culture Collection (ATCC). HaCaT cells were purchased from AddexBio Technologies. 293TT cells were generated by introducing SV40 Large T antigen cDNA into HEK293T cells to increase Large T antigen expression and obtained from Christopher Buck (NIH). All cell lines were cultured at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20 mM HEPES, 10% fetal bovine serum (FBS), L-glutamine, and 100 units/mL penicillin streptomycin (DMEM10). Cell identity was confirmed by ATCC cell authentication service.


Although Rab9a is required for efficient infection by HPV pseudovirus (PsV) [12,15], its role in this process has not been investigated. To determine the role of Rab9a in HPV infection, HeLa S3 cervical cancer cells were transfected with non-targeting control siRNA (siNC) and two different siRNAs targeting Rab9a expression (siRab9a and siRNA9a-2). Rab9a knockdown was confirmed by Western blotting (Figs 1A and S1A). These cells were then infected with HPV16 PsV consisting of a complete L1 and L2 capsid containing a reporter plasmid expressing GFP. A 3xFLAG tag was appended to the C-terminus of L2. Infectivity was determined by flow cytometry for GFP fluorescence at 48 h post infection (hpi).


Rab GTPases play key regulatory roles in intracellular vesicular trafficking [1,2,4–6]. Like other small GTPases, Rab proteins exist in two forms, an active GTP-bound state necessary for trafficking of cellular cargo and an inactive GDP-bound state [1,3]. Accordingly, GTP-Rab9a supports the transport of cellular cargos such as CI-MPR and DMT1-II (Fig 6) [36,37]. Surprisingly, however, we show here that HPV trafficking during entry is impaired by excess GTP-Rab9a and stimulated by excess GDP-Rab9a (Figs 5A–5E and S9). These findings indicate that HPV and cellular proteins utilize the Rab9a host trafficking machinery in distinct ways during intracellular trafficking (Fig 7A).


We would like to thank Qun Lin for making HPV18 and HPV5 PsVs. We also thank Changin Oh for providing plasmid DNAs of p16SheLL-HA WT and 3R mutant. We also thank Yuka Takeo and Jian Xie for their technical advice for performing proximity ligation assay and co-immunoprecipitation analysis. Flow Cytometry was conducted in the flow cytometry shared resource of the Yale Cancer Center.

Citation: Choi J, DiMaio D (2023) Noncanonical Rab9a action supports retromer-mediated endosomal exit of human papillomavirus during virus entry. PLoS Pathog 19(9): e1011648.

Editor: Craig Meyers, Penn State University School of Medicine, UNITED STATES

Received: April 26, 2023; Accepted: August 29, 2023; Published: September 13, 2023

Copyright: © 2023 Choi, DiMaio. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by a grant from the National Cancer Institute to DD (R35-CA242462) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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