Ultrashort pulsed laser treatment inactivates viruses by inhibiting viral replication and transcription in the host nucleus

Abstract

Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated. This information is critical for the optimization and the extension of this treatment platform to other applications. Toward this goal, we investigated whether viral internalization, replication, or gene expression in cells were altered by ultrashort pulsed laser irradiation. Murine Cytomegalovirus (MCMV), an enveloped DNA virus, was used as a model virus. Using electron and fluorescence microscopy, we found that laser-treated MCMV virions successfully internalized in cells, as evidenced by the detection of intracellular virions, which was confirmed by the detection of intracellular viral DNA via PCR. Although the viral DNA itself remained polymerase-amplifiable after laser treatment, no viral replication or gene expression was observed in cells infected with laser-treated virus. These results, along with evidence from previous studies, support a model whereby the laser treatment stabilizes the capsid, which inhibits capsid uncoating within cells. By targeting the mechanical properties of viral capsids, ultrashort pulsed laser treatment represents a unique potential strategy to overcome viral mutational escape, with implications for combatting emerging or drug-resistant pathogens.

Keywords: Murine cytomegalovirus; Pathogen inactivation; Pathogen reduction; Ultrashort pulsed lasers.

Figure 1. Proposed model for USP laser-induced protein aggregation

USP laser light scattering leads to transient, partial unfolding of protein structures, due to disruption of electrostatic interactions through ISRS. In cases where the protein is at high density, aggregation will occur between exposed hydrophobic patches on nearby proteins. In cases where the protein is at low density, the proteins rapidly recover by regaining their native conformation. Filled spheres indicate hydrophilic regions of the protein; hollow spheres indicate hydrophobic regions of the protein.

Figure 2. Electron microscopy shows cellular internalization of USP laser-treated MCMV

Control or laser-treated MCMV were infected into murine embryonic fibroblast cells, and the cells were harvested, fixed, and sectioned for imaging. Images show cellular internalization of control MCMV or USP laser-treated MCMV at 2h post-infection. Arrows indicate intracellular virions.

Figure 3. Fluorescence microscopy shows cellular internalization of USP laser-treated MCMV

PKH26-labeled control or laser-treated MCMV were infected into murine embryonic fibroblast cells. Images show combined bright field and fluorescence depicting cellular internalization of control MCMV or USP laser-treated MCMV at 2h post-infection.

Figure 4. MCMV DNA is present in cells infected with USP laser-treated MCMV

Murine embryonic fibroblast cells were infected with control or laser-treated MCMV. PCR analysis was performed at 18h post-infection with primers specific for the MCMV IE1 gene. M: molecular weight DNA ladder; 1: cells infected with control MCMV; 2: cells infected with laser-treated MCMV.

Figure 5. USP laser-treated MCMV cannot replicate in cells

Control GFP-expressing MCMV or laser-treated GFP-expressing MCMV virus were infected into murine embryonic fibroblast cells and imaged for GFP expression 24, 48, and 72h post-infection using fluorescence microscopy. Images show GFP expression in cells infected with either control MCMV or laser-treated MCMV at 24, 48, and 72h post-infection. BF, bright field; GFP, green fluorescent protein.

Figure 6. IE1 protein is absent in cells infected with USP laser-treated MCMV

Murine embryonic fibroblasts were either mock-infected or infected with control (untreated) MCMV or laser-treated MCMV. At the time points after infection indicated, cells were harvested and lysed, subjected to SDS-PAGE electrophoresis analysis, and immunoblotted with anti-IE1 or anti-actin monoclonal antibodies.

Figure 7. DNA from USP laser-treated MCMV virions is PCR-amplifiable

Control or laser-treated MCMV virions were subjected to PCR analysis with primers specific for the MCMV IE1 gene. M: molecular weight DNA marker; 1: control MCMV; 2: laser-treated MCMV; 3: control MCMV pre-digested with proteinase K; 4: laser-treated MCMV pre-digested with proteinase K.

Figure 8. Proposed model for USP laser-induced viral capsid defect

USP laser treatment causes aggregation of viral capsid proteins. The laser-treated virus is internalized by cells, but the aggregated viral capsid cannot uncoat and thus the viral genome remains “trapped” and cannot replicate or express viral genes.