Human pDCs preferentially sense enveloped hepatitis A virions
Zongdi Feng, … , Christopher M. Walker, Stanley M. Lemon
Zongdi Feng, … , Christopher M. Walker, Stanley M. Lemon
Published November 21, 2014
Citation Information: J Clin Invest. 2015;125(1):169-176. https://doi.org/10.1172/JCI77527.
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Research Article Virology

Human pDCs preferentially sense enveloped hepatitis A virions

Abstract

Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-α via TLR7 signaling when cocultured with infected cells. pDCs required either close contact with infected cells or exposure to concentrated culture supernatants for IFN-α production. In isopycnic and rate-zonal gradients, pDC-activating material cosedimented with eHAV but not membrane-bound acetylcholinesterase, suggesting that eHAV, and not viral RNA exosomes, is responsible for IFN-α induction. pDC activation did not require virus replication and was associated with efficient eHAV uptake, which was facilitated by phosphatidylserine receptors on pDCs. In chimpanzees, pDCs were transiently recruited to the liver early in infection, during or shortly before maximal intrahepatic IFN-stimulated gene expression, but disappeared prior to inflammation onset. Our data reveal that, while membrane envelopment protects HAV against neutralizing antibody, it also facilitates an early but limited detection of HAV infection by pDCs.

Authors

Zongdi Feng, You Li, Kevin L. McKnight, Lucinda Hensley, Robert E. Lanford, Christopher M. Walker, Stanley M. Lemon

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Figure 2

eHAV induces IFN-α production by pDCs.

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eHAV induces IFN-α production by pDCs. (A) Serial dilutions of concentra...
(A) Serial dilutions of concentrated supernatant (100,000-g pellet) from HAV-infected cells were mixed with pDCs (1 × 106/ml) and incubated for 20 hours. IFN-α levels (mean ± range in replicate assays) are plotted against HAV RNA content. Unconcentrated supernatant from infected (red arrow and triangle) and mock-infected (white square) cells were tested in parallel. (B) Concentrated supernatant fluids from HAV-infected cells were subjected to isopycnic gradient centrifugation, and individual gradient fractions were incubated with pDCs (1 × 106/ml). HAV RNA content of fractions was determined by RT-qPCR. IFN-α production shown represents results from 3 donors (mean ± SEM). AChE activity was measured by enzyme assay. (C) Correlation (Spearman’s test) between eHAV content of isopycnic gradient fractions, shown as genome equivalent per pDC, and IFN-α produced (mean ± range in replicate assays). (D) Northern blot of HAV RNA: full-length in vitro–transcribed HM175/18f HAV RNA, RNA extracted from peak isopycnic gradient fractions containing eHAV, or nonenveloped HAV (mean ± range in replicate assays). (E) Consecutive gradient fractions containing eHAV from a gradient similar to that in B were pooled, concentrated, and subjected to rate-zonal ultracentrifugation. Fractions were collected from the top and assessed for HAV RNA content, AChE activity, and pDC stimulating activity (P105 and P106 indicate individual donors) (mean ± range in replicate assays). Representative results from 1 of 3 independent experiments are shown. Western blots of ALIX and flotillin-1 in the rate-zonal gradient fractions are shown.