No reservations: sequestration of resources during Pseuorabies virus superinfection exclusion

Abstract

Pseudorabies Virus (PRV) is an alphaherpesvirus, exhibiting neurological infection and latency in the natural porcine host, similar to the related human-infecting Herpes simplex viruses. Typical of many viruses, PRV's preferred entry mechanism is pH-dependent endocytosis. Once PRV establishes infection, it will prevent subsequent virions from initiating a secondary infection within infected cells--a phenomenon known as superinfection exclusion (SIE). SIE has been hypothesized to be a means of sequestering cellular resources and limiting genetic drift, while also influencing viral replication and evolution. Previous work from our laboratory has shown that PRV-induced exclusion prevents secondary virions from entering host cells, but whether those virions are trapped at the cell surface or within an early endosome near the surface remains unknown. We hypothesize that PRV impairs cellular endocytosis during exclusion, preventing secondary virion entry. Using fluorescently labelled fluid-phase markers, we found that PRV infection does not cause a global shutdown of endocytosis, but instead induces the formation of large, stalled, endosomal compartments. From these observations, we propose that PRV SIE may be specific to incoming virion cargo rather than a global shutdown of all endocytic pathways. We also found that fluorescently labelled dextran and wheat germ agglutinin (WGA) uptake revealed persistent aggregates in PRV-infected cells, distinct from late endosomal compartments marked by LAMP-1. These phenotypes were mimicked in mock- infected cells treated with alkalinizing agents, suggesting impaired endosomal acidification. Together, these findings hint that early PRV-induced changes to endosomal trafficking may prime the host endosomal landscape for subsequent stages of infection, such as immune-evasion and or secondary envelopment.