Microbial pathogens rely on their host cells to replicate, and thus have evolved a multitude of mechanisms to exploit host cell machinery, divert cellular resources, and remodel cells to form a favorable environment for replication. One global target of microbial pathogens is host actin, a highly conserved protein that is involved in a myriad of cellular functions. Actin is found in the cell in two forms, monomeric G-actin and filamentous F-actin, and is a major component of the eukaryotic actin cytoskeleton. A notable group of pathogens that target host actin are baculoviruses, which are large enveloped DNA viruses that replicate in host cell nuclei. Baculoviruses represent an extreme example of actin subversion by a pathogen, as they use actin at almost every step of the viral replication cycle. Specifically, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is unique among viruses in its extraordinary augmentation of actin within the nucleus during infection. In particular, the AcMNPV protein AC102 is essential for viral replication and facilitates the nuclear localization of G-actin (termed NLA) during early AcMNPV infection. However, AC102 shares no significant primary sequence similarity with non-baculovirus proteins and has no recognizable sequence motifs, making it difficult to predict how AC102 functions in NLA or what other processes it may be involved in during AcMNPV infection.
Through my dissertation research, I have provided a basic characterization of AC102’s expression profile, localization, and protein interactions. My research also reveals several previously unknown roles of AC102 in late stage AcMNPV infection processes. First, my research shows that AC102 is expressed predominantly during late infection. This result was surprising given that the only previously known function of AC102 was during early infection. AC102’s late expression suggested that AC102 may also have role(s) during late infection. To further investigate AC102’s function(s), I generated ten mutant viruses, each containing a single point mutation in a conserved amino acid residue of AC102, and screened for viruses with replication defects. While most of these viruses exhibited modest 2- to 5-fold reductions in viral titer compared with wild type virus, the AC102-K66A mutant virus produced 10-fold fewer progeny, and was therefore chosen for further analysis.
Characterization of the AC102-K66A mutant virus revealed many defects in late stage viral infection, indicating that AC102 does indeed play an important role in late viral functions. First, the AC102-K66A mutant virus is defective in nucleocapsid morphogenesis, as the major capsid protein VP39 forms long tubular filaments that are often bundled together in AC102-K66A infected cells instead of properly packaged nucleocapsids. This indicates that AC102 is important for proper nucleocapsid assembly during late infection. Second, the AC102-K66A mutant virus is defective in the assembly of F-actin in the nucleus during late infection. Nuclear F-actin polymerization is mediated through the actions of viral protein P78/83 (an actin nucleation promoting factor) and the host Arp2/3 actin nucleating complex. Interestingly, a tagged version of wildtype AC102 co-purifies with a previously identified nucleocapsid-associated protein complex consisting of viral proteins P78/83, C42, and EC27. This indicates that AC102 is a previously unidentified member of the P78/83-C42-EC27-AC102 complex. AC102’s interaction with P78/83 in a nucleocapsid-associated protein complex provides an explanation for AC102’s roles in nucleocapsid morphogenesis and nuclear F-actin polymerization. Future research will reveal how AC102 mechanistically contributes to the functions of the P78/83-C42-EC27-AC102 complex and if there is a regulatory link between AC102’s roles in early NLA and late nuclear F-actin polymerization.
