Rhinoviruses are the major causative agents of the common cold and cost the United States economy approximately $40 billion per year (6
). Therefore, it is of great interest to prevent or ameliorate the symptoms of the common cold. The rhinovirus genus is a member of the picornavirus family and is characterized by nonenveloped capsid with a diameter of ~300 Å containing a single-stranded, plus-sense RNA genome (19
). Other members of the picornavirus family include foot-and-mouth disease virus, poliovirus, encephalomyocarditis virus, and hepatitis A virus. The capsids exhibit pseudo T = 3 icosahedral symmetry and are composed of 60 copies of the four capsid proteins VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 have an eight-stranded antiparallel beta-barrel motif structure and form the outer surface of the capsid, while VP4 lies at the interface between the capsid and the interior genomic RNA (22
). VP4 is approximately 70 amino acids in length and is myristoylated at the N terminus (3
Antibodies are the major line of defense against picornavirus infections. In the case of human rhinovirus 14 (HRV14), a number of studies have been performed to detail the antibody recognition and neutralization processes (25
). While it had been long suggested that antibodies neutralize viral infectivity by inducing large conformational changes in the capsid, both cryo-transmission electron microscopy (cryo-TEM) (2
) and crystallographic analysis (27
) clearly demonstrated that this was not the case. Further, it was shown that antibody recognition is more plastic than previously thought in that it is able to bind into the relatively narrow receptor-binding region of the canyon (27
). These results suggested that the major in vivo role of antibodies is to bind to virion and work synergistically with other immune system components (26
). This hypothesis has gained further support from studies of other pathogens (1
) and implies that vaccines need only to elicit antibodies that bind to the authentic pathogen with high affinity.
While these results simplified the goal of creating a synthetic vaccine by focusing on capsid recognition rather than possible antibody-induced conformational changes, developing synthetic vaccines against all 100 serotypes of HRV remains a daunting task. As shown in the structures of HRV14/antibody complexes, the antibodies make extensive contacts with the surface of the capsid that is not limited to a single antigenic loop (2
). Further evidence for this extensive contact is that antibodies to peptides corresponding to antigenic NIm loops fail to neutralize the virions (17
), and antibodies raised against intact capsids do not bind effectively to peptides corresponding to NIm-IA loop (T. J. Smith, unpublished results). One notable exception is the case of HRV2, where there is cross-reactivity between the NIm-II site of the virion and a synthetic peptide (30
). Nevertheless, developing a repertoire of peptides representing the entire antigenic ensemble of HRVs is not only impractical but also unlikely to elicit neutralizing antibodies.
All of the studies described above were performed with the antibodies that were raised against intact particles or to peptides representing epitopes that reside on the outer surface of the capsid. In the case of poliovirus, however, antibodies were raised against VP4 and the N termini of VP1 of poliovirus serotype I (15
). It was shown that these antibodies are capable of neutralizing the virion despite the fact that those portions of the capsid protein are buried in the interior of the capsid at the capsid-RNA interface (8
). These results suggested that the poliovirus capsid was more dynamic than indicated by the crystal structure and that these termini are presented to the exterior of the virion in a temperature-dependent and reversible manner. While the role of capsid dynamics in the viral life cycle was not clear, it was suggested that the N termini of VP1 and VP4 might facilitate cell membrane attachment and subsequent entry of the virus into the host cell (3
More recently, evidence for capsid dynamics has been found in other viruses as well. In the cases of swine vesicular disease virus (10
) and coxsackievirus A9 (18
), antibodies were raised against the whole virus in pigs and rabbits, respectively. These polyclonal antibodies demonstrated a strong reaction to the peptides corresponding to the N termini of VP1 and VP3 of swine vesicular disease virus and coxsackievirus A9, respectively. In a similar study, antibodies from the plasma of patients suffering from type I diabetes were found to target VP4 protein of coxsackievirus B3, again suggesting the exposure of VP4 peptide during coxsackievirus infection (23
). These results imply that capsid “breathing” may be a phenomenon common to many proteinaceous capsids.
Using a very different approach, the dynamic nature of HRV14 was analyzed using limited proteolysis and mass spectrometry (matrix-assisted laser desorption ionization [MALDI]) analyses (14
). In these experiments, the virus was treated with both matrix-bound and soluble forms of trypsin for various periods of time, and the resulting proteolytic fragments were identified by MALDI. Surprisingly, the N termini of VP4 and VP1 were found to be the most proteolytically sensitive portions of the capsid in spite of being buried inside the viral capsid. As an additional control, the antiviral “WIN” compounds, which had been previously shown to stabilize the virions against thermal and acid denaturation, were added during digestion. While these WIN compounds did not affect the intrinsic proteolytic activity of trypsin, they nearly completely protected the VP1 and VP4 termini from proteolysis for an extended period. Together, these results suggested that HRV14 is transiently exposing these termini in a “breathing” process and that the empty hydrophobic drug-binding region apparently plays an important role in facilitating these dynamics.
In this study we further examined HRV14 capsid dynamics by raising polyclonal antibodies against several peptides representing the N termini of VP1 and VP4. In these experiments, only the antibodies against the VP4 N terminus were found to successfully neutralize viral infectivity in vitro. Further, we demonstrate that the HRV14 VP4 antiserum cross-reacts with other serotypes of rhinovirus (HRV16, and HRV29), which is likely due to the high degree of conservation of VP4. Antibody neutralization closely parallels the MALDI analysis in that antibody neutralization and proteolysis are enhanced at 37°C in the case of HRV16 whereas the elevated temperatures are not required for either phenomenon in the cases of HRV14 and HRV29. Epitope mapping of the N-terminal 30 residues of VP4 suggests that it adopts a nonlinear conformation, and this is further substantiated by results showing that all of the copies of VP4 in the Ser5Cys HRV14 mutant at room temperature form cysteine cross-linked dimers. This cysteine cross-link does not form at 4°C, suggesting that capsid breathing is essential for VP4 exposure and interactions. Since VP4 dimerization does not affect viral infectivity, it seems likely that VP4 extrusion is a normal part of the cell attachment and entry process of rhinovirus. Together, these results suggest that VP4 might be useful as a pan-serotypic rhinovirus vaccine, but it seems likely that better understanding of the VP4 oligomeric structure will be necessary for further optimization.