Biology subjects

Zimina, A.

Publications and source records attributed to Zimina, A..

3 recordsLinked to original sources

bioRxiv2023

The Development of Resistance to an Inhibitor of a Cellular Protein Reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.

microbiology↗

bioRxiv2022

A Proximity Biotinylation Assay with a Host Protein Bait Reveals Multiple Factors Modulating Enterovirus Replication

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, arguing that the GBF1 environment is linked to the viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified those previously associated with the enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins many of which normally localize in the nucleus constituted the largest group, underscoring the massive release of nuclear factors in the cytoplasm of infected cells and their involvement in the viral replication. Analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly increase the knowledge about the organization of enterovirus replication organelles and may provide new targets for anti-viral interventions.

microbiology↗

bioRxiv2022

Characterization of enterovirus D68 infection in four nonhuman primate species

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host specific factors that predict disease severity and progression to the neurologic syndrome. Published animal models of EV-D68 infection to date have been limited to mice, cotton rat and ferrets, and investigation of the susceptibility of nonhuman primate (NHP) species to contemporary EV-D68 isolates has not yet been reported. In this study, we challenged juvenile NHPs - cynomolgus macaques, rhesus macaques, pigtailed macaques, and African green monkeys - with one of five different 2014 or 2018 EV-D68 isolates by the respiratory route. Animals were monitored for clinical respiratory and neurologic signs, and serially collected nasal swabs, bronchoalveolar lavage fluid (BALF) and cerebrospinal fluid (CSF) were evaluated for EV-D68 RNA and infectious virus. Infection with 2014 and 2018 EV-D68 isolates resulted in mild respiratory and gastrointestinal disease in some animals, but no evidence of neurological disease. Neither EV-D68 RNA nor infectious virus could be detected from any sample collected from animals challenged with 2014 EV-D68 isolates. Limited viral shedding - based on viral RNA quantified from nasal swabs and BALF - was detected from some animals infected with 2018 EV-D68 isolates. No virus was detectable in CSF. The rate of seroconversion was 100% for cynomolgus macaques infected with the 2018 EV-D68 isolates, but averaged between 0-50% for the 2014 isolates. Based on the results of this study, there is some evidence that infection with 2018 EV-D68 isolates may be more reliable at establishing limited infection than 2014 EV-D68 isolates. Regardless of virus isolate, however, EV-D68 infection of juvenile NHP species resulted in mild and nonspecific clinical disease and limited viral shedding. These data suggest that further refinements to the NHP model system (e.g., immunosuppression and/or direct viral inoculation) may be required to reproduce EV-D68 infection of the central nervous system and the associated AFM phenotype.

microbiology↗