Antiviral agent blocks breathing of the common cold virus

Abstract

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a ''breathing'' model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.Human rhinovirus 14 (HRV14), the causative agent of the common cold, is a member of a family of animal viruses-the picornaviruses-whose other members include the polio, hepatitis A, and foot-and-mouth disease viruses (1). The HRV14 virion consists of an icosahedral protein shell, or viral capsid, surrounding an RNA core. The capsid is made up of 60 copies of each of four structural proteins, VP1-VP4. Based on crystal structure data (2), VP1, VP2, and VP3 compose the viral surface, whereas VP4 lies interior at the capsid͞RNA interface. To examine the solution structure of HRV14, we used protein mass mapping, limited proteolysis with mass spectrometry (3, 4). Here the site-specific proteolytic degradation of a protein results in a set of digestion fragments which are subsequently mass analyzed by matrix-assisted laser desorption͞ionization mass spectrometry (MALDI-MS). The resulting digestion fragments provide structural information concerning the individual capsid proteins as well as their proteinprotein interactions, because available cleavage sites are dependent on both the tertiary and quaternary protein structure. For instance, cleavage sites residing on the exterior of the virus will be most accessible to the enzyme and therefore be among the first digestion fr...

Rhinovirus infections and immunisation induce cross-serotype reactive antibodies to VP1

“…Most antibodies generated to RV infection in humans are directed against epitopes found on surface-exposed structures of VP1, VP2 and VP3 (Carey et al, 1992). Although there is evidence that the N-terminus of VP4 may be transiently exposed at the capsid surface in a process known as capsid breathing (Lewis et al, 1998) and antibodies to this region of VP4 are cross-serotype protective (Katpally et al, 2009), naturally occurring human antibodies to VP4 have not been described. A recent study by Niespodziana et al revealed that children with RV-induced respiratory symptoms primarily generate IgG1 and IgA to N-terminal regions of VP1 and that responses to VP2, VP3 and VP4 were significantly lower .…”

Viral capsid mobility: A dynamic conduit for inactivation

“…One mechanism involves the modification of proteins, which would cause inhibition of viral cell entry or the release of the genome. The second mechanism allows the alkylating reagents direct access to the viral genome through a mobile protein capsid (8)(9)(10)(11)(12). The recent findings (8)(9)(10)(11) that the protein capsids of viruses in solution have a much higher degree of dynamics than their crystallized counterparts suggested that the second mechanism might be the means of inactivation.…”

“…The second mechanism allows the alkylating reagents direct access to the viral genome through a mobile protein capsid (8)(9)(10)(11)(12). The recent findings (8)(9)(10)(11) that the protein capsids of viruses in solution have a much higher degree of dynamics than their crystallized counterparts suggested that the second mechanism might be the means of inactivation. Focusing on this latter idea, the ability of small alkylating agents to react with either the capsid or encapsidated nucleic acid was investigated initially by using flock house virus (FHV), an RNA-containing model virus used in previous studies (8,9).…”

The Application of Prophylactic Antibodies for Rhinovirus Infections

“…A further complication for RVs is that the aforementioned NIms are largely serotype specific and that highly conserved capsid epitopes are not normally surface exposed and therefore unavailable for conventional Ab binding. Nevertheless, sequence conservation is found at the N terminus of the buried capsid protein VP4, which is transiently exposed at the surface in a process known as capsid breathing [5], and polyclonal Abs to this region are cross-serotype protective in vitro [14]. Such rare Abs may be worth further investigation as potential prophylactics, as are those Abs with the ability to interact with the conserved residues found deep in the capsid canyon -the site of ICAM-1 binding for major group RVs.…”

“…Nevertheless, several Abs (approximately 10 of 200) are undergoing preclinical development or are in clinical trials for viral infectious diseases targets including HIV-1, HBV, human rhinoviruses (RV) and human cytomegalovirus [3]. RV may be particularly suited to the application of prophylactic Abs as no appropriate antiviral therapies are available despite numerous attempts [5][6][7], vaccine development faces challenging obstacles [8,9] and new information regarding protective antibody responses is being unearthed [10][11][12][13][14]. Thus, a full evaluation of the application of prophylactic Abs to these important human pathogens where there is an explicit need for appropriate therapeutic/ prophylactic interventions is warranted.…”