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.

Figure 1

Trypsin digestion time course of HRV14 (Inset). The asymmetric unit of HRV14 is shown such that the interior RNA is located at the bottom of the diagram, the nearest 5-fold axis on the left, and the nearest 2-fold axis on the right. VP1, VP2, VP3, and VP4 are blue, green, red, and mauve, respectively. Large yellow balls denote initial cleavage sites after a 5-min incubation with trypsin. Doubly charged species in the mass spectra are denoted by 2+. T = 0 represents undigested virus with VP4 observed at m/z= 7,390.0; VP1–VP3 (not shown) are observed at m/z = 32,518.9 (VP1 expected 32, 519.5 Da), 28,475.9 (VP2 expected 28, 477.4 Da), and 26,219.5 (VP3 expected 26, 217.8 Da).

Figure 2

X-ray crystal structure of HRV14 shows VP4 to be the most internal viral protein. Time-course mass mapping of HRV14, however, reveals proteolytic fragments originating from VP4 within the first few minutes of viral digestion, demonstrating transient exposure of VP4 to the viral surface. The large arrow illustrates the translocation of VP4 from the interior of the capsid to the external viral surface (the arrow does not indicate the mechanism of the translocation). Also shown are the position of naturally occurring escape mutation sites for NIm-IA, NIm-IB, NIm-II, and NIm-III in yellow, black, cyan, and white, respectively.

Figure 3

HRV14 digestion with and without Win 52084 drug present. The MALDI-MS mass analysis was performed on HRV14 alone before digestion (A), after digestion of the HRV14/drug mixture (B), and after digestion of HRV14 alone (no drug present) (C). Digestions shown were performed for 3 h. It is important to note that in each case the virus was exposed for the same amount of time and the same concentration of enzyme; yet the drug-exposed virus was extremely resistant to digestion. Control experiments with FHV clearly demonstrated that the drug was not inhibiting the enzyme.

Figure 4

Control experiment. Trypsin digestion of FHV (5) in the presence and absence of Win 52084. The off scale peak is the FHV γ-peptide (m/z 4,397 Da). Comparison of the resulting spectra shows the two sets of digestion fragments to be identical (within normal fluctuations of MALDI-MS signal intensity) and illustrates that inhibition of HRV14 digestion in the presence of the drug is a result of the HRV14-drug interaction and not Win 52084 inhibition of trypsin.