Superinfection Exclusion in Alphabaculovirus Infections Is Concomitant with Actin Reorganization

UNLABELLED: Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.