A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Abstract

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virus-specific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses ('eHAV') resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between 'enveloped' and 'non-enveloped' viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

Figure 1. Enveloped particles (eHAV) are the dominant form of virus released from infected cell cultures

a, HAV genome organization. The polyprotein is depicted as a box with pX highlighted. b, Buoyant density of HAV particles released by Huh-7.5 cells in iodixanol gradients. eHAV bands at 1.06–1.10 g/cm³, whereas nonenveloped HAV bands at 1.22–1.28 g/cm³. GE, genome equivalents. c, (left) HAV capsid antigen was detected by ELISA only in pools of denser fractions from the gradient in b. (right) Capsid antigen was detected in light fractions after treatment with 1% NP-40. Data shown are mean OD₄₅₀ ± range in duplicate assays. d, Electron microscopic images of negative-stained eHAV (i-iv from fraction 10 in b) and a nonenveloped virion (v, fraction 20 in b). e, Infectious titer of pooled fractions containing eHAV or nonenveloped virions before and after chloroform extraction. FFU, focus-forming units. f, Specific infectivity of pooled fractions containing eHAV or nonenveloped virions, calculated by dividing the HAV RNA copy number (GE, qRT-PCR) by infectious titer (FFU, IR-FIFA). Values shown are means ± range from duplicate RT-PCR reactions. g, eHAV is resistant to neutralization by anti-capsid monoclonal antibody K24F2. Antibody-virus mixtures were incubated for 1 hr at 37 °C, inoculated onto cells for 1 hr, followed by removal of the inoculum, washing ×3 with PBS, and addition of an agarose overlay. Viral antigen was visualized by infra-red immunofluorescence (IR-FIFA). h, Immunoblots of HAV capsid proteins (VP1 and VP2) in lysates of mock or HAV-infected cells (lanes 1 and 2), gradient-purified eHAV (lane 3), and chloroform-extracted nonenveloped virions (lane 4).

Figure 2. eHAV circulates in the blood of HAV-infected humans and chimpanzees

a, Distribution of HAV RNA in an iodixanol gradient loaded with early, acute-phase serum from patient BH12. b, Buoyant density of HAV particles from plasma and feces of an experimentally-infected chimpanzee, ×0293⁵. Fecal HAV RNA is ×10⁵, while plasma RNA is ×10² (also see Supplementary Fig. 4). The low buoyant density of circulating virus was not due to a passive effect of serum, as the buoyant density of nonenveloped virions was not altered by suspension in 90% serum (data not shown).

Figure 3. eHAV biogenesis requires ESCRT-associated proteins

a, siRNA knockdown of ESCRT-associated proteins. Immunoblots of HRS, TSG101, VPS4B, and ALIX in HAV-infected cells 72 hrs after transfection with the indicated gene-specific targeting (T) or non-targeting (NT) control siRNAs. (*), nonspecific protein band. qRT-PCR assays confirmed >80% knockdown of TSG101 and VPS4B (Supplementary Fig. 6c). b, Total viral RNA detected in culture fluids 48–72 hrs after siRNA transfection. c, Relative yield and buoyant density of virus released from siRNA transfected cells (as in panel a) determined in iodixanol gradients. RNA associated with eHAV particles (fractions 8–12) was reduced 85% and 87% by depletion of VPS4B and ALIX, respectively. d, One-step growth of eHAV in cells transfected with NT and gene-specific siRNAs. Huh7.5 cells were transfected with indicated siRNA for 3 days, then infected with gradient-purified eHAV at a multiplicity of infection (m.o.i.) of 20. Cell-associated HAV RNA was assayed by qRT-PCR at the times indicated. Knockdown efficiency was confirmed at the end of the experiment (not shown). e, Tandem YPX₃L ALIX-interacting motifs (red typeface) in VP2. Below are the motif sequences in wild-type (wt, HM175/18f) and mutant viral RNAs. f, Total viral RNA released into culture fluids 24–48 hrs after electroporation of Huh-7.5 cells with wild-type and mutant viral RNAs. Δ3D is a replication-incompetent subgenomic replicon RNA with a lethal frame-shift mutation in 3D^pol. g, Intracellular HAV RNA following electroporation of cells with wild-type and VP2 mutant RNAs. h, Confocal microscopy showing K24F2 detection of capsid antigen in cells electroporated 48 hrs previously with wild-type or mutant RNAs. K24F2 recognizes a conformation-dependent assembled neutralization epitope in the viral capsid. i, Anti-capsid (K24F2) and anti-ALIX (Bethyl) antibody-mediated immunoprecipitation of encapsidated, RNase-resistant viral RNA in detergent-treated lysates of cells electroporated with wild-type or mutant HAV RNAs. RT-PCR data represent mean ± s.e.m. from 2–3 replicate assays; all results are representative of 2–3 independent experiments.

Figure 4. Extracellular eHAV is resistant to antibody-mediated neutralization, but is neutralized by antibody following infection

a, Neutralization of eHAV in cells treated with anti-HAV before (pre) or after (post) adsorption of virus. Pre-treated cells were incubated with JC plasma (1:50–1:100) for 1 hr, washed extensively with PBS, then incubated with gradient-purified eHAV (m.o.i. ~6) for 1 hr in the absence of antibody. For post-treatment, JC was added to the medium upon refeeding after removal of the inoculum. Cells were subsequently refed at 24 hr intervals with fresh media containing no antibody. Data shown represent means ± s.e.m. from duplicate cultures, and are representative of 6 independent experiments. b, Adding anti-HAV to medium as late as 6 hrs after removal of the inoculum restricts the replication of eHAV (left panel), but not nonenveloped HAV (right panel). JC antibody was added at intervals after removal of the inoculum, and intracellular viral RNA quantified at 48 hrs. Results shown are mean ± s.e.m. of replicate RT-PCR assays and representative of 3 independent experiments. c, Chloroquine (50 µM) blocks eHAV but not HAV entry. Cells were treated for 30 min prior to inoculating virus, and harvested at 48 hr to assay intracellular HAV RNA. d, Anti-TIM-1 mAb 190-4 blocks entry of both eHAV and HAV. GL37 cells were incubated with 190-4 (10–100 µg/ml) or control IgG (100 µg/ml) for 1 hr at 37 °C, then inoculated with virus for 1 hr. Intracellular HAV RNA was assayed at 72 hrs. RT-PCR data represent mean ± s.e.m. from 2–3 replicate assays; all results are representative of 2–3 independent experiments.