The Rhinovirus subviral a-particle exposes 3'-terminal sequences of its genomic RNA

Abstract

Enteroviruses, which represent a large genus within the family Picornaviridae, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (+)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that in vitro acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ∼700 bases. However, even after hours of incubation at pH 5.2, 5'-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment in vivo. This finding suggests that the exposed 3'-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded.

Importance: For host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification in vitro results in RNA exit starting from the 3'-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.

FIG 1

Sequences at the 3′ end of the viral RNA, including the poly(A) tail, become accessible upon acidification of native HRV-A2 at 34°C. Normalized autocorrelation curves of A) DyLight 488-labeled oligo(dT) incubated with native virus (black) and with virus incubated at pH 5.2 for 15 min (red) and for 60 min (blue). (B) Same as in panel A, but in the presence of an oligonucleotide complementary to a sequence close to the 5′ end (positions 443 to 468). The diagrams show the components present in the incubation mixtures and their interactions. Note the absence of free RNA and of sequences recognized by the 5′-specific oligonucleotide at all time points.

FIG 2

RNA sequences externalized on incubation of HRV-A2 at pH 5.2 are not recognized by dsRNA-specific MAb J2, as shown by capillary electrophoresis. Acidified or native virus, as indicated, were incubated with the specified components and analyzed by capillary electrophoresis. The subviral particle exposing RNA (“intermediate particle” [I]) migrates as a broad peak at a position different from that of the A-particle (see also reference 27). Upon removal of the exposed RNA sequences with MNase, the particle comigrates with the A-particle. Note that the addition of poly(U) together with MAb J2, but neither one alone, modified the migration of intermediate particles. Note that the replacement of MAb J2 with an irrelevant antibody (anti-γ-tubulin) at the same concentration had no effect on the migration of viral or subviral particles. Finally, note that native virus did not change its migration upon the addition of poly(U) together with MAb J2, demonstrating the absence of accessible poly(A) sequences. Internal standard (is), dimethyl sulfoxide (DMSO). Broken vertical lines indicate the position of the “intermediate particles.” Electropherograms were calibrated to the migration of DMSO and, where present, of RNasin (R). N, native virus; A, A-particle.

FIG 3

At pH 5.2, HRV-A2 releases an RNA segment, including about 700 residues at the 3′ end. (A) Diagram illustrating the procedure. Virus was incubated in acidic buffer at 34°C for the times indicated, the buffer was reneutralized, and the accessible RNA was digested. (B) Native HRV-A2 (control) and the subviral particles resulting from this treatment were separated on a 0.7% agarose gel. RNA within the particles was visualized with GelGreen. DNA markers were run in the leftmost lane. (C) RNA was extracted from the bands in panel B, and the presence of sequences was assessed with RT-qPCR using primer pairs hybridizing to the indicated positions in the viral genome. The number of copies is given as an arbitrary number. The approximate trend of the time-dependent loss of RNA sequences (from 3′ to 5′) is indicated by a trend plot.

FIG 4

In vivo release of viral RNA is rapid. HeLa cells (10⁶) grown in suspension culture were suspended in 1 ml of infection medium, and HRV-A2 (multiplicity of infection of 1,000) was allowed to attach at 4°C for 4 h. The cells were then washed with PBS, resuspended in fresh infection medium, and incubated for the indicated times. Next, the cells were lysed, intermediate particles were collected via immunoprecipitation with 2G2, externalized RNA was digested with MNase, and protected sequences were identified with RT-PCR using the primer pairs complementary to the viral genome at the positions indicated at the left. In the upper right panel (control), uncoating was inhibited via addition of niclosamide (Nic), present during the whole experiment, and immunoprecipitation was performed with polyclonal rabbit antiserum directed against HRV-A2 (αHRV-A2). Experiments were carried out at the temperatures indicated under otherwise identical conditions. The status of the virion and of its RNA is shown as a diagram below the gels; black line, time of conversion into A-particle; blue line, time until the 3′ end becomes accessible from the A-particle; green line, time until complete RNA release. N, native; A, A-particle; I, intermediate particle (with partially released RNA); E, empty capsid.

FIG 5

HeLa microsomal fraction promotes RNA uncoating. HRV-A2 was acidified in the presence of subcellular fractions (left) or of microsomal fractions obtained from a sucrose density step gradient (right). For the upper panel, subviral particles were immunoprecipitated with MAb 2G2, and RNA was extracted from the immunoprecipitates and analyzed for the presence of 5′-proximal sequences via RT-PCR using primer pair M, followed by 1% agarose gel electrophoresis and GelGreen staining. To exclude loss of the RNA via digestion by contaminating RNases, the supernatant was again immunoprecipitated, but now with MAb 8F5 (66) to remove any remaining native virus. RNA was then extracted from the supernatant and analyzed as described above. As a control, an irrelevant RNA (ARA; Arabidopsis mRNA containing ubiquitin-encoding sequences) was added to all samples prior to immunoprecipitation and RNA extraction and was detected with a ubiquitin-specific primer set. Fractions framed were positive for LAMP1 and EEA1, as determined via Western blotting (data not shown). Lower bands are from the primers. To exclude contamination of the reaction mixture with viral RNA, RT-PCR was carried out without the addition of viral material (negative control). The results from one of five independent experiments are shown.