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. 2020 Jun 16;94(13):e00367-20.
doi: 10.1128/JVI.00367-20. Print 2020 Jun 16.

hnRNPA2B1 Associated with Recruitment of RNA into Exosomes Plays a Key Role in Herpes Simplex Virus 1 Release from Infected Cells

Xusha Zhou  1 , Lei Wang  1 , Weixuan Zou  1 , Xiaoqing Chen  2 , Bernard Roizman  3   4 , Grace Guoying Zhou  5   2
Affiliations

hnRNPA2B1 Associated with Recruitment of RNA into Exosomes Plays a Key Role in Herpes Simplex Virus 1 Release from Infected Cells

Xusha Zhou et al. J Virol. .

Abstract

hnRNPA2B1, an abundant cellular protein, has been reported to recruit RNAs bearing a specific sequence (EXO motif) into exosomes. We characterized an exosome population averaging 100 ± 50 nm in diameter and containing a defined set of constitutive exosome markers. This population packages microRNAs (miRNAs) and can be directed to block targeted gene expression in a dose-dependent fashion. The objective of this study was to characterize the role of hnRNPA2B1 in the recruitment of miRNA. We report the following four key findings. (i) hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. Hence, hnRNPA2B1 carries its cargo, at most, to the site of exosome assembly, but it is not itself incorporated into exosomes. (ii) The accumulation of exosomes produced by cells in which the gene encoding hnRNPA2B1 has been knocked out (ΔhnRNPA2B1 cells) was reduced 3-fold. (iii) In uninfected HEp-2 cells, hnRNPA2B1 is localized in the nucleus. In cells infected with herpes simplex virus 1 (HSV-1), hnRNPA2B1 was quantitatively exported to the cytoplasm and at least a fraction of hnRNPA2B1 colocalized with a Golgi marker. (iv) Lastly, in ΔhnRNPA2B1 cells, there was a 2- to 3-fold reduction in virus yield but a significant (>10-fold) reduction in HSV-1 released through the apical surface into the extracellular environment. The absence of hnRNPA2B1 had no significant impact on the basolateral export of HSV-1 from infected to uninfected cells by direct cell-to-cell contact. The results suggest that hnRNPA2B1 plays a key role in the transport of enveloped virus from its site of assembly to the extracellular environment.IMPORTANCE In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells.

Keywords: HSV-1; exosome; hnRNPA2B1; release.

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Figures

FIG 1
FIG 1
(A) Validation of hnRNPA2B1-knockout cell line. HEp-2 cells or ΔhnRNPA2B1 cells in a 12-well plate were harvested, solubilized, subjected to electrophoresis in a 10% denaturing gel, and treated with antibodies against hnRNPA2B1 or GAPDH. (B) Particle size distribution and number of isolated exosomes extracted from ΔhnRNPA2B1, parental HEp-2, and HEK293T cells. Exosomes were isolated from HEK293T, HEp-2, or ΔhnRNPA2B1 cells as described in Materials and Methods. Particle size distribution and the number of isolated exosomes were analyzed by Izon’s qNano technology.
FIG 2
FIG 2
Quantification of exosomes purified from HEK293T, parental HEp-2, and ΔhnRNPA2B1 cells. HEK293T, HEp-2, or ΔhnRNPA2B1 cultures each containing 5 × 106 cells were rinsed with PBS and incubated in serum-free medium for 24 h. The exosomes were isolated from culture medium as described in Materials and Methods. Exosomes purified from equal amounts of cells and 10 μg of cell pellets were analyzed for the presence of exosomal marker proteins (CD9, annexin V, flotillin-1, Alix, calnexin, CD63, hnRNPA2B1, and GAPDH) by immunoblotting as detailed in Materials and Methods. Calnexin was used as a negative exosome marker protein.
FIG 3
FIG 3
Intracellular localization of Golgi marker protein TGN46 and hnRNPA2B1. HEp-2 cells were mock infected or exposed to 10 PFU of HSV-1 per cell for 1 h. The inoculum was replaced with fresh culture medium. The cells were fixed at the times shown and were costained with an antibody to TGN46 (green, a trans-Golgi marker protein) as well as those to hnRNPA2B1 (red) and DAPI (blue, for nuclei). The images were captured and processed using a confocal laser-scanning microscope (magnification, ×63 and ×200).
FIG 4
FIG 4
Accumulation of intracellular and extracellular virus in HEp-2 and ΔhnRNPA2B1 cell cultures exposed to 10 PFU of virus per cell. The virus progeny in the cell pellet and medium were harvested at indicated time points, and the titers were determined in Vero cells.
FIG 5
FIG 5
Photographs (A and B) and average plaque size (C) of plaques produced by HSV-1 in HEp-2 and ΔhnRNPA2B1 cell cultures. HEp-2 cells and ΔhnRNPA2B1 cells grown in T25 flasks were exposed to 0.01 PFU of HSV-1(F) and then overlaid with DMEM supplemented with 1% FBS plus 0.05% (wt/vol) immunoglobulin. At 72 h postinfection, the cells were fixed with 4% (wt/vol) paraformaldehyde for 30 min, stained with Giemsa for 30 min, and photographed at ×20 magnification with the aid of an inverted microscope. The size of the plaques formed by HSV-1 in HEp-2 and ΔhnRNPA2B1 cell cultures was calculated as a percentage of the plaque size in HEp-2 cells.
FIG 6
FIG 6
(A) Accumulation of viral proteins in HEp-2 cells and ΔhnRNPA2/B1 cells. HEp-2 or ΔhnRNPA2/B1 cells were mock infected or infected with one PFU/cell of HSV-1(F) for 24 h. The cell lysates were harvested, electrophoretically separated in a 10% denaturing gel, and treated with indicated antibodies. (B) The efficiency of hnRNPA2B1-specific siRNA. HEp-2 cells were transfected with nontarget (NT) siRNA or siRNA targeting hnRNPA2B1 (Si-hnRNP-1 and Si-hnRNP-2). After 72 h of incubation, the cells were harvested, electrophoretically separated in a 10% denaturing gel, and treated with indicated antibodies. (C) The accumulation of viral proteins in HEp-2 cells transfected with the hnRNPA2B1-specific siRNA. HEp-2 cells were transfected with nontarget (NT) siRNA or Si-hnRNP-2 siRNA. After 72 h of incubation, the cells were mock infected or infected with 0.1 PFU/cell for 24 or 36 h. At indicated times, the cell lysates were harvested, electrophoretically separated in a 10% denaturing gel, and treated with indicated antibodies.
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