I always tell my students that any stage of virus replication can be a target for antiviral therapy – as long as it is essential to replication and specific to the virus and therefore that inhibiting it does not damage the host cell. So far we have mostly limited ourselves to a very few stages of the replication cycle, and we only have one or two drugs (against influenza virus) that inhibit the vital uncoating step of the replication cycle. Understanding the processes involved is therefore of great importance in developing new drugs. A recent paper in PLOS Pathogens examines the uncoating of rhinovirus particles and makes some interesting findings.
Human rhinoviruses (HRV) are members of the picornavirus family, and are one of the major causative agents of the common cold. Additionally, they play important roles in the exacerbation of asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Similar to other picornaviruses, the rhinovirus particle are composed of 60 copies each of four capsid proteins, VP1, VP2, VP3 and VP4, arranged on an icosahedral lattice. The diameter of the particle is roughly 30 nm. The virus genome is a single-stranded RNA molecule of positive sense, about 7100 bases in length. It carries a covalently linked peptide (VPg) at its 5′-end and a poly-(A) tail of about 70 to 150 bases at its 3′-end. The 5′-nontranslated region is approximately 650 bases in length, highly structured, and involved in cap-independent translation initiation and RNA replication.
Minor group rhinoviruses, e.g. the prototype strain HRV2, bind members of the low-density lipoprotein receptor (LDLR) family including LDLR and LDLR-related protein for entry via clathrin-dependent endocytosis. Once in the endosome, the low pH leads to dissociation of the virus from the receptors as well as to structural changes in the viral capsid. The native virion sedimenting at 150S converts into the subviral A-particle sedimenting at 135S and devoid of the internal capsid protein VP4 and exposure of amphipathic N-terminal sequences of VP1 renders it hydrophobic, thus allowing its direct attachment to the inner endosomal membrane. These processes are accompanied by an expansion of the virus shell by about 4% along with the opening of symmetry-related channels. The largest channels are at the two-fold axes, whereas the smaller ones are located near the pseudo three-fold axes and at the base of the star-shaped vertices of the icosahedron. Finally, the RNA is released through one of these pores, most probably at a 2-fold axis. The final product of this uncoating process is the empty capsid (80S B-particle). Most enteroviruses undergo similar conformational changes; however, with the exception of minor receptor group rhinoviruses, the process appears to be triggered by receptor binding and possibly assisted by low pH.
These structural modifications can be mimicked, at least partially, in vitro. Exposure to pH <5.8 converts native HRV2 preferentially into A-particles whereas incubation at 50°C–56°C in low ionic strength buffers favours conversion into B-particles (empty capsids). In vivo, and in the presence of liposomes in vitro, both VP4 and N-terminal sequences of VP1 insert into lipid bilayers. They might contribute to formation of a pore connecting the virus interior with the cytosol of the host cell, thus allowing for the transit of RNA in its unfolded form.
The mechanism of RNA exit is poorly understood. Energy would be required for breaking the hydrogen bonds of the double-stranded regions in the encapsidated RNA genome in order to allow the RNA to thread through an opening only large enough to enable passage of a single strand. It appears likely that either the poly-(A) tail at the 3′-end or the VPg peptide linked to the 5 end of the RNA begins to emerge from the virion since other modes might be unproductive (e.g., simultaneous exit of both ends would be expected to impede complete uncoating and thus to be abortive). Directionality of this process may indicate that the RNA adopts a defined conformation inside the viral shell suggesting a well-organized process of assembly and uncoating.
The new paper shows that RNA exit does indeed occur in a specific and ordered manner, starting from the 3′-end. Ordered exit of RNA also suggests that the virus genome becomes organized during packaging or assembly, which may occur co-transcriptionally. Therefore, it is likely that the process of encapsidation begins when the 5′-end emerges from the replication complex or at least before the complete RNA has been synthesized. It is also possible that the same applies to other viruses with ssRNA of positive polarity. This would imply that in these viruses, the 3′-end becomes encapsidated last, remaining near the capsid wall presumably in close proximity to one of the holes poised to open upon uncoating, thus resulting in a ‘last-in-first-out’ process of assembly and uncoating.
So all we need now is a drug to inhibit this process, and we’ve cured the common cold. Well, some of them maybe :-)
Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End. (2013) PLoS Pathog 9(4): e1003270. doi:10.1371/journal.ppat.1003270
Viral infection requires safe transfer of the viral genome from within the protective protein shell into the host cell’s cytosol. For many viruses this happens after uptake into endosomes, where receptor-binding and/or the acidic pH trigger conformational modifications or disassembly of the shell, allowing the nucleic acids to escape. For example, common cold viruses are converted into subviral particles still containing the single-stranded positive sense RNA genome; subsequently, the RNA escapes into the cytoplasm, leaving behind empty capsids. We triggered this process by heating HRV2 to 56°C and found that 3′- and 5′-end emerged with different kinetics. Crosslinking prevented complete RNA egress and upon nuclease digestion only sequences derived from the 5′-end were protected. Part of the RNA remaining within the viral shell adopted a rod-like shape pointing towards one of the two-fold axes where the RNA is presumed to exit in single-stranded form. Egress thus commences with the poly-(A) tail and not with the genome-linked peptide VPg. This suggests that assembly and uncoating are well-coordinated to avoid tangling, kinetic traps, and/or simultaneous exit of the two RNA ends at different sites.