In this study, we found that reovirus colocalizes with microtubule tracks during cell entry and requires microtubule function and microtubule motor dynein 1 to efficiently traverse the endocytic pathway. Microtubule function is not required for internalization of reovirus virions but rather facilitates targeting of reovirus to acidified endosomes for viral disassembly. Treatment of cells with microtubule inhibitors blocks reovirus infection in a temporal window in which the virus transits from early to late endosomes. These results highlight a new function for microtubules in reovirus replication and suggest that impairment of microtubule activity might diminish infection by viruses that require access to late endosomes to establish productive infection.
Endocytic uptake of macromolecular cargo requires the coordinated action of several host factors, including receptors, Rab GTPases, and enzymes that regulate endocytic transport by modifying targets at specific intracellular sites. For some cargo, microtubules and microtubule-associated motors are required for transport to and from the cell surface. Importantly, the maturation of early to late endosomes is dependent on microtubule function (34
). Rab-interacting lysosomal protein (RILP), a Rab7 adapter, recruits dynactin and dynein to late endosomes, promoting late endosome movement toward the cell interior (35
). Reovirus is transported to Rab7-marked endosomes during cell entry (13
), and expression of dominant-negative RILP inhibits reovirus infection (13
). Thus, disruption of microtubule function appears to delay reovirus disassembly by slowing the maturation of the reovirus-containing endosomal fraction. This model is supported by the observed decrease in colocalization of reovirus with LAMP1-marked endosomes. Additional support comes from the observation that vindesine sulfate does not diminish infection after adsorption of ISVPs, which are uncoated in vitro
and thus do not require access to cathepsin-containing organelles to establish infection (16
). As an important control, vindesine sulfate does not inhibit infection by CHKV, which uncoats in early endosomes (33
). Impairment of endosomal maturation might be responsible for the aggregates of reovirus particles observed in vindesine sulfate-treated cells. Interestingly, reovirus virions do not accumulate in early endosomes in cells treated with vindesine sulfate (). Instead, viral particles in vindesine sulfate-treated cells aggregate in clusters in the cytoplasm. These observations suggest that in the absence of microtubule function, viral particles are missorted during cell entry and not trapped in early endosomes.
Our report highlights the potential for drugs that inhibit endosomal maturation for use as broadly active anti-infectives. Such drugs could inhibit viruses, bacteria, bacterial toxins, and parasites that require access to late endosomes and lysosomes to mediate pathological effects. Avian reovirus, a fusogenic reovirus that, unlike mammalian reovirus, enters cells via caveolin-1 and causes infected cells to form syncytia (36
), also uses microtubules to enter cells (36
). This finding suggests that employment of microtubules by fusogenic and nonfusogenic reoviruses is a conserved cell entry mechanism despite the use of different endocytic uptake pathways. Adenovirus (38
) and Borna disease virus (39
) also use microtubules and microtubule motors during cell entry (40
). Enterococcus faecalis
requires microtubules for efficient internalization into cells (41
). Cytotoxic necrotizing factor 1 (CNF1), a toxin produced by some pathogenic Escherichia coli
strains, requires microtubule function to access late endosomes for the processing required for cytotoxicity (42
). Flubendazole, a compound identified in our screen as impairing reovirus cytotoxicity, inhibits infection by nematodes (43
). While currently available microtubule-inhibiting compounds are associated with significant adverse effects (21
), it is possible that safer agents could be developed for anti-infective therapies that transiently inhibit endosomal maturation.
Reovirus strain T3D, which has been trademarked as Reolysin, is being evaluated in clinical trials for efficacy as an oncolytic agent in combination with various chemotherapeutic drugs, including the microtubule inhibitor docetaxel (5
). Our findings suggest that pairing reovirus with microtubule-inhibiting agents during oncolytic therapy may limit virus-induced cell killing. Cancer treatment regimens that use reovirus and microtubule-inhibiting drugs may be more efficacious if the administration of virus and chemotherapeutic is not simultaneous. We found that addition of vindesine sulfate to reovirus-infected cells at up to 1 h after infection fails to significantly diminish infection. Thus, we think it important to assess the effects of pharmacological agents on viral infectivity when these treatments are used in combination.
The NCC screen yielded six candidate compounds that do not target microtubules. Procarbazine, which promotes DNA damage (44
), and 6-azauridine, which inhibits pyrimidine synthesis (45
), likely impair reovirus replication by affecting viral transcription or genome replication. The identification of nicotinic acetylcholine receptor and serotonin receptor agonists as drugs that impair reovirus-induced cytotoxicity points to interesting cellular targets. The nicotinic acetylcholine receptor is expressed in the brain (46
), and serotonin receptors are expressed in both the brain and gastrointestinal tract (47
). Both of these organs are sites for reovirus replication in the infected host (1
). Finally, the identification of indomethacin, which inhibits cyclooxygenase 1 and 2 (48
), suggests a yet-uncharacterized function for cyclooxygenases in reovirus replication. Further studies are required to determine whether these drugs inhibit reovirus replication and to define the antiviral mechanisms by which they act.
The identification of host molecules that regulate steps in viral replication enhances an understanding of how viruses use basic cellular processes to propagate and disseminate. These studies also yield new knowledge about cellular functions and illuminate new targets for antiviral drug development. In this study, we used a high-throughput screening approach to identify microtubules and microtubule motor dynein 1 as host factors required for reovirus cell entry, initiation of infection, and consequent cell death. Findings made in this study should contribute to the development of improved strategies for use of reovirus as an oncolytic and establish a platform for testing microtubule inhibitors as anti-infective agents.