Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport

Abstract

Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

FIG. 1.

9C12 binding does not inhibit HAdV-mediated endosomalysis. (A) 9C12 binding does not stabilize the virus capsid for vertex dissociation. Capsid-associated proteins from Ad5.eGFP samples incubated at the indicated temperatures in the presence of the neutralizing 9C12 antihexon antibody or a control, nonneutralizing antihexon antibody were analyzed by SDS-PAGE and Simply Blue staining. Bands corresponding to hexon (H), penton base (PB), fiber (F), and proteins IIIa, V, VI, and VII are indicated. Note the equivalent loss of vertex proteins (bold). A labeled molecular mass standard is in the left lane. Data are representative of three independent experiments. (B) [³⁵S]Met incorporation into cellular proteins was measured as a function of Ad5.eGFP concentration upon infection of A549 cells in the presence or absence of the neutralizing 9C12 antihexon antibody or a nonneutralizing antihexon antibody (control) and the ribotoxin α-sarcin. The ts1 mutant of HAdV-2 (ts1) was included as a control. Data are the mean percentages of [³⁵S]Met incorporation compared to control uninfected cells for triplicate samples ± standard deviations and are representative of three experiments.

FIG. 2.

9C12 binding blocks accumulation of HAdV-5 at the nuclear envelope. (A) Representative A549 cells at the indicated times postinfection with Ad5.eGFP in the presence or absence of the neutralizing 9C12 antibody stained for tubulin (FITC, green), Ad5.eGFP (Cy3, red), and the nucleus (DAPI, blue) are depicted as z-profiles of sequential deconvolved z-series. Bars, 10 μm. Percentages are the colocalization values for each cell. (B) Colocalization of Ad5.eGFP with the nucleus (DAPI) was quantified. Data are the mean percent colocalization ± the standard error of the mean for an average of 60 cells per data point.

FIG. 3.

9C12 binding increases the association of HAdV-5 with microtubules in the presence and absence of microtubule-associated proteins. Ad5.eGFP binding to microtubules (MTs) was measured in the presence or absence of the neutralizing 9C12 antihexon antibody or a control, nonneutralizing antihexon antibody. Virus was incubated with MTs alone or in the presence of a MAPF with or without 500 mM NaCl. Data are the fold increase of bound hexon for each sample compared to the no-antibody sample incubated with MTs and MAPF without NaCl (*, positive control) ± the standard deviation for three independent experiments. Bands are fluorescent scans of bound and 8% of input Cy3-labeled hexon from a representative experiment.

FIG. 4.

9C12 binding prevents HAdV-5 from reaching the microtubule-organizing center. (A) Representative A549 cells at 90 min p.i. with Ad5.eGFP in the presence or absence of the neutralizing 9C12 antibody and the CRM1 inhibitor LMB stained for tubulin (FITC, green), Ad5.eGFP (Cy3, red), and the nucleus (DAPI, blue) are depicted as z-profiles of sequential deconvolved z-series. Bars, 10 μm. Percentages are the colocalization values for each cell. (B) Colocalization of Ad5.eGFP with the nucleus (DAPI) was quantified. Data are the mean percent colocalization ± standard error of the mean for an average of 80 cells per data point.

FIG. 5.

9C12 does not directly block hexon binding to the nuclear envelope. Accumulation of Alexa Fluor 488-labeled HAdV-5 hexon at the nuclear envelope of digitonin-permeabilized NRK cells was visualized in the presence or absence of the neutralizing 9C12 antihexon antibody or a control, nonneutralizing antihexon antibody. Cells incubated with the nuclear import factor importin β (Impβ) were included as a positive control for blocking hexon binding to the nuclear envelope. Individual z-sections of cells stained with DAPI to identify the nucleus are shown and are representative of at least three experiments.