Rhinovirus‐stabilizing activity of artificial VLDL‐receptor variants defines a new mechanism for virus neutralization by soluble receptors

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Minor group human rhinoviruses (HRVs) bind three members of the low-density lipoprotein receptor (LDLR) family: LDLR proper, very-LDLR (VLDLR) and LDLR-related protein (LRP). Whereas ICAM-1, the receptor of major group HRVs actively contributes to viral uncoating, LDLRs are rather considered passive vehicles for cargo delivery to the low-pH environment of endosomes. Since the Tyr-Trp-Thr-Asp ␤-propeller domain of LDLR has been shown to be involved in the dissociation of bound LDL via intramolecular competition at low pH, we studied whether it also plays a role in HRV infection. Human cell lines deficient in LDLR family proteins are not available. Therefore, we used CHO-ldla7 cells that lack endogenous LDLR. These were stably transfected to express either wild-type (wt) human LDLR or a mutant with a deletion of the ␤-propeller. When HRV2 was attached to the propeller-negative LDLR, a lower pH was required for conversion to subviral particles than when attached to wt LDLR. This indicates that high-avidity receptor binding maintains the virus in its native conformation. HRV2 internalization directed the mutant LDLR but not wt LDLR to lysosomes, resulting in reduced plasma membrane expression of propeller-negative LDLR. Infection assays using a CHO-adapted HRV2 variant showed a delay in intracellular viral conversion and de novo viral synthesis in cells expressing the truncated LDLR. Our data indicate that the ␤-propeller attenuates the virus-stabilizing effect of LDLR binding and thereby facilitates RNA release from endosomes, resulting in the enhancement of infection. This is a nice example of a virus exploiting high-avidity multimodule receptor binding with an intrinsic release mechanism.

Section: Resultssupporting

confidence: 82%

Section: Discussionmentioning

confidence: 88%

Low pH-Triggered Beta-Propeller Switch of the Low-Density Lipoprotein Receptor Assists Rhinovirus Infection

Konecsni

Berka

Pickl‐Herk

et al. 2009

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“…Nanocontainers purified by spin SEC were decorated with His 6 -tagged MBP-V33333 via the DOGS-NTA incorporated into the lipid membrane to allow for subsequent HRV2 binding (nanocontainer/receptor/virus [NCϩRϩV] complexes). Since the multivalent receptor impedes movements of the capsid proteins with respect to each other (19,29), HRV2-receptor complexes require a lower pH for conversion into subviral particles than free virus. Therefore, in order to keep the concentration of free receptor low (to avoid formation of virus-receptor complexes in solution), it was used at a molar ratio of about 0.01 with respect to Ni 2ϩ -NTA groups (assuming that 50% of the NTA groups are found on the inner leaflet).…”

Section: Resultsmentioning

confidence: 99%

“…Under the same conditions, but using liposomes without receptor, similar leakage effects were observed (data not shown). As stated above, the lack of a stimulating effect of the receptor might be explained by the multivalent nature of the binding impeding movements of viral capsid proteins with respect to each other (29), thus inhibiting the structural changes necessary for exposure of amphipathic domains. The stabilization of the virus might thus counteract the virus-concentrating effect of the receptor and explain the Ϫ6 (Ͻ10 virions/ nanocontainer); at higher ratios the cDNA signal decreases again.…”

Section: ϫ5mentioning

confidence: 99%

Liposomal Nanocontainers as Models for Viral Infection: Monitoring Viral Genomic RNA Transfer through Lipid Membranes

Bilek

Matscheko²,

Pickl‐Herk³

et al. 2011

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After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

“…At least in the case of minor group HRVs, it cannot be explained by a "catalytic" function of the receptor; binding of a very-low density lipoprotein receptor mimetic, a head-to-tail concatemer of ligand-binding repeat V3 that exhibits high affinity for HRV-A2 (61) stabilizes the virus rather than aiding in its conversion to A-particles (62). This is dissimilar to major group viruses which are uncoated by an excess of soluble ICAM-1 alone (63).…”

Section: Discussionmentioning

confidence: 99%

The Rhinovirus Subviral A-Particle Exposes 3′-Terminal Sequences of Its Genomic RNA

Harutyunyan

Kowalski

Blaas

2014

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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 ϳ700 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. IMPORTANCEFor 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.