Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1–dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N 1 -acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo. These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCERNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target. P olyamines are small, positively charged molecules, derived from arginine, that are involved in several cellular processes in mammalian and nonmammalian cells. Studies carried out on herpesviruses demonstrated that viral capsids contain significant amounts of polyamines that putatively neutralize charges on the viral DNA in order to facilitate compaction and encapsidation (1). Vaccinia virus (2) and bacteriophage R17 (3) also incorporate polyamines into virions. We recently demonstrated that chikungunya virus (CHIKV) and Zika virus (ZIKV), important pathogens responsible for serious outbreaks, rely on polyamines for both translation and transcription (4). The potential role of polyamines in virus replication and the possibility of targeting the polyamine biosynthetic pathway for more diverse array of RNA viruses, including those involved in outbreaks, has not been examined.Several drugs have been developed that can target the polyamine biosynthetic pathway. Perhaps the best known inhibitor is difluoromethylornithine (DFMO; also called eflornithine), an FDA-approved drug that is used to treat trypanosomiasis (5-7) and hirsutism (8), as wel...

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7/ensconsin family proteins. We found that all four mammalian MAP7 family members bound to kinesin-1, and MAP7, MAP7D1 and MAP7D3 acted redundantly to enable kinesin-1-dependent transport in HeLa cells. Microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions, was inhibited in cells co-depleted of these three MAP7 proteins. In vitro, purified MAP7 and MAP7D3 increased microtubule landing rate and processivity of KIF5B-560.The same was true for MAP7D3 C-terminus, which weakly bound to microtubules and exchanged rapidly on motile KIF5B-560 motors. A C-terminal MAP7 fragment lacking microtubule affinity increased KIF5B-560 recruitment to microtubules in vitro and in cells, and partially rescued kinesin-1-dependent transport in the absence of full-length MAP7 proteins. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules. SummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.

Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden.IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.