Inactivation of enveloped virus by laser-driven protein aggregation

Abstract

Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction.

Fig. 1

Experimental setup for femtosecond laser irradiation. An 850 nm (near-infrared) laser beam was frequency doubled to produce 425 nm (visible) irradiation. Samples were magnetically stirred to expose the virus to the laser-focused volume.

Fig. 2

Inactivation of MCMV using a femtosecond laser. Femtosecond laser-induced reduction in viral titer was assessed by TCID assay. Results are representative of quadruplicate experiments and error bars indicate SEM.

Fig. 3

Preservation of MCMV global virion structure after femtosecond laser irradiation. Representative electron microscopy images are shown of control and laser-irradiated virions (150 to 200 nm in diameter) at both $\mathrm{50,000}\times$ and $\mathrm{150,000}\times$ magnification, showing no clear differences in the global appearance of viral envelope and capsid structures after irradiation.

Fig. 4

Preservation of MCMV genomic DNA integrity after femtosecond laser irradiation. Agarose gel analysis was performed on genomic DNA isolated from control or irradiated MCMV, showing essentially identical banding patterns, which suggests a lack of double-strand breaks or crosslinking of viral DNA after femtosecond laser irradiation. DNA was electrophoresed intact or after digestion using restriction enzymes. For clarity, image contrast and sharpness were enhanced uniformly across the entire image using Photoshop. Results are representative of at least two experiments.

Fig. 5

Aggregation of MCMV virion proteins after femtosecond laser irradiation. SDS-PAGE analysis was performed on control and laser-irradiated virions. The arrow denotes a high molecular weight aggregate formed by proteins in the laser-treated group. The aggregate was excised for mass spectrometry analysis. For clarity, image contrast and sharpness were enhanced uniformly across the entire image using Photoshop. Results are representative of at least two experiments.