Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles

Abstract

Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the U37, ICP0, and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity compared to their corresponding counterparts containing low VP16 or VP22 content. These findings were corroborated for VP16 in short interfering RNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is influenced not only by the genome of viruses but also by the stoichiometry of proteins within each virion. The ability of viruses to spread in animals has been mapped to several viral genes, but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts, while others vary substantially. Interestingly, this correlates with the VP16 -activating viral protein and indirectly with VP22, a second virion component whose modulation profoundly alters virion composition. This reaffirms that not only the presence but also the amount of specific tegument proteins is an important determinant of viral fitness.

Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy

“…The goodness of fit to a normal distribution of VP5 intensities exceeded 0.95 with a CV of 23.75 (S3 Fig). This variance in particle intensity is very similar to analogous types of HSV single particle analysis using either GFP-fusion proteins or antibody to capsid protein [32,33]. With this as a benchmark, we found the distribution genome signal detected by cycloaddition to have only marginally increased variance with a CV of 34.19 and a goodness of fit to normal of 0.94 (S3 Fig).…”

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity

“…The deletion of UL49, coding for VP22 in pseudorabies virus (PRV), has only a minor impact on viral growth (14,15), whereas in HSV-1, the lack of VP22 is rapidly compensated for by mutations in UL41 (16)(17)(18). However, HSV-1 null mutants replicate less efficiently and do not accumulate at the cell surface, and the absence of VP22 affects the virion composition and indirectly modulates viral fitness (19)(20)(21).…”

“…Additional experiments would be required to verify whether some other viral proteins are mislocalized or incorrectly expressed in VZV-ORF9-L231-V5-infected cells and whether the final composition of the released virions is affected. A very elegant flow virometry analysis of the extracellular HSV-1 particles indicates that the particles sorted for their high VP22 content show a modest but reproducible increase in infectivity compared to the particles containing smaller amounts of VP22, although the authors concluded that the VP22 level acts only indirectly on viral fitness (20). Although technically difficult due to the small amount of particles produced, a mass spectrometry analysis of VZV-ORF9-L231A-V5 particles would certainly be informative.…”

Flow Virometry: a Powerful Tool To Functionally Characterize Viruses

“…Despite these exciting initial reports, the analysis of viruses by flow cytometry, which Grivel and colleagues termed "flow virometry" in 2013 (14), has become more mainstream only recently. Flow virometry has now been used to characterize an expanding array of additional viruses, including lambda phage (15), herpes simplex virus 1 (HSV-1) (16,17), mouse hepatitis virus (MHV) (18), human immunodeficiency virus (HIV) (14,(19)(20)(21), Nipah virus (22), Junin virus (23), vaccinia virus (24), dengue virus (20,25), human cytomegalovirus (HCMV) (26), and giant viruses (13). Flow virometry is becoming a powerful tool to characterize viruses.…”