Inactivation of respiratory syncytial virus by zinc finger reactive compounds

Abstract

Background: Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins.

Results: Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease.

Conclusions: This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.

Figure 1

Inactivation of respiratory syncytial virus by AT-2, Tetramethylthiuram disulfide, and Tetraethylthiuram disulfide. RSV was treated with 10 mM of the indicated compounds for 24 hrs at 37°C. Viral titers were then determined by plaque assay on HEp-2 cells. Control reactions (corresponding to "0 mM" of each chemical) contained the same amount of DMSO as that present in drug-containing incubations.

Figure 2

Inability of respiratory syncytial virus inactivated with AT-2 to replicate in the lungs of cotton rats. RSV was inactivated by incubation with 10 mM AT-2 for 24 hrs at 37°C. Virus was then diluted with PBS and used to intranasally infect cotton rats. Control animals received sham-treated RSV. Four days post-challenge animals were sacrificed and lungs were collected for viral titrations. Results shown are the mean log (viral titer) ± SE for 4 animals per group.

Figure 3

Time- and dose-dependence of RSV inactivation by AT-2. (A) Time course of RSV inactivation. RSV was incubated with 10 mM, 30 mM AT-2 or the corresponding amount of DMSO (control, no AT-2) at 37°C for different times. At the indicated time points, reaction was stopped and viral infectivity was determined by plaque assay on HEp-2 cells. Dashed line indicates the limit of the sensitivity of titration assay. (B) Dose response curve of RSV inactivation by AT-2. RSV was incubated with the indicated amounts of AT-2 at 37°C for 24 hrs. Viral titers were determined by plaque assay on HEp-2 cells. Dashed line indicates the limit of the sensitivity of titration assay.

Figure 4

Respiratory syncytial virus inactivation by AT-2 is accompanied by modification of the M2-1 protein. RSV was inactivated by incubation with 10 mM AT-2 for 24 hrs at 37°C (lanes 2 and 4), or with corresponding amount of DMSO (lanes 1 and 3). At the end of the incubation, RSV particles were lysed and analyzed by SDS-PAGE either in the presence (lanes 3 and 4), or in the absence (lanes 1 and 2) of β-ME, followed by immunoblotting with IgY against RSV: (A) M2-1 protein, (B) P protein, and (C) N protein.

Figure 5

Immunogenicity of AT-2-inactivated RSV in the cotton rat model. Cotton rats were immunized intramuscularly with AT-2-inactivated RSV ("RSV/AT-2"), AT-2-inactivated RSV adjuvanted with Ribi emulsion (adjRSV/AT-2) or formalin-inactivated RSV (FI-RSV). Control animals remained unvaccinated ("control") or were immunized via repeated infection with live RSV ("live RSV"). All animals were challenged with RSV and sacrificed 4 days after infection for analysis of pulmonary viral load (A) and histopathology (B). Results are the mean ± SEM for 4 animals per group. *p < 0.05 when compared to the "control" group.