Biology subjects

Mladinich, M. C.

Publications and source records attributed to Mladinich, M. C..

3 recordsLinked to original sources

bioRxiv2023

ZIKV Induction of Tristetraprolin in Endothelial and Sertoli Cells Post-Transcriptionally Inhibits IFNβ/{lambda} Expression and Promotes ZIKV Persistence

Zika virus (ZIKV) is a mosquito-borne Flavivirus that persistently infects patients, enters protected brain, placental, and testicular compartments, is sexually transmitted, and causes fetal microcephaly in utero. ZIKV persistently infects brain microvascular endothelial cells (hBMECs) that form the blood-brain-barrier and Sertoli cells that form testicular barriers, establishing reservoirs that enable viral dissemination. ZIKV persistence requires inhibiting interferon (IFN) responses that direct viral clearance. We found that ZIKV induces IFN-{beta} and IFN-{lambda} in hBMECs but post-transcriptionally inhibits IFN-{beta}/{lambda} expression. IFN{beta}/{lambda} mRNAs contain AU-rich elements (AREs) in their 3 untranslated regions which regulate protein expression through interactions with ARE binding proteins (ARE-BPs). We found that ZIKV infection of primary hBMECs induces the expression of the ARE-BP tristetraprolin (TTP) and that TTP is a novel regulator of endothelial IFN secretion. In hBMECs, TTP knockout (KO) increased IFN-{beta}/{lambda}1 mRNA abundance and IFN-{beta}/{lambda}1 secretion in response to ZIKV infection and inhibited viral persistence. In contrast, TTP expression dramatically reduced IFN-{beta}/{lambda}1 secretion in hBMECs. IFN-{beta}/{lambda}1 mRNA stability was not significantly altered by TTP and is consistent with TTP inhibition of IFN-{beta}/{lambda}1 translation. TTP is similarly induced by ZIKV infection of Sertoli cells, and like hBMECs, TTP expression or KO inhibited or enhanced IFN-{beta}/{lambda} mRNA levels, respectively. These findings reveal a mechanism for ZIKV induced TTP to promote viral persistence in hBMECs and Sertoli cells by post-transcriptionally regulating IFN-{beta}/{lambda} secretion. Our results demonstrate a novel role for virally induced TTP in regulating IFN secretion in barrier cells that normally restrict viral persistence and spread to protected compartments. ImportanceOur findings define a novel role for ZIKV induced TTP expression in regulating IFN-{beta}/{lambda} production in primary hBMECs and Sertoli cells. These cells comprise key physiological barriers subverted by ZIKV to access brain and testicular compartments and serve as reservoirs for persistent replication and dissemination. We demonstrate for the first time that the ARE binding protein TTP is virally induced and post-transcriptionally regulates IFN-{beta}/{lambda} secretion. In ZIKV infected hBMEC and Sertoli cells, TTP knockout increased IFN-{beta}/{lambda} secretion, while TTP expression blocked IFN-{beta}/{lambda} secretion. The TTP directed blockade of IFN secretion permits ZIKV spread and persistence in hBMECs and Sertoli cells and may similarly augment ZIKV spread across IFN-{lambda} protected placental barriers. Our work highlights the importance of post-transcriptional ZIKV regulation of IFN expression and secretion in cells that regulate viral access to protected compartments and defines a novel mechanism of ZIKV regulated IFN responses which facilitate neurovirulence and sexual transmission.

microbiology↗

bioRxiv2023

Establishment of a CPER Reverse Genetics System for Powassan Virus Defines Attenuating NS1 Glycosylation Sites and an Infectious NS1-GFP11 Reporter Virus

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long term neurologic damage. Currently there are no POWV therapeutics, licensed vaccines or reverse genetics systems for producing infectious POWVs from recombinant DNA. Here we used a circular polymerase extension reaction (CPER) approach to generate recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. Flavivirus NS1 proteins are highly conserved glycoproteins that regulate replication, spread and neurovirulence. POWV NS1 proteins contain three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated with a 1-2 log decrease in infectious titers. The severely attenuated recLI9-N224Q mutant exhibited a 2-3 day delay in focal cell-to-cell spread and reduced NS1 secretion. Like WT LI9, the recLI9-N224Q mutant was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations attenuate POWV neuroinvasion in vivo. To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1 lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly and secretion, and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo, and enables the rational genetic design of live attenuated POWV vaccines.

microbiology↗

bioRxiv2021

Powassan Viruses Spread Cell to Cell During Direct Isolation from IxodesTicks and Persistently Infect Human Brain Endothelial Cells and Pericytes

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the Northeastern U.S., with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted in as little as 15 minutes of a tick bite, and enter the CNS to cause encephalitis (10% fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of spread cell to cell, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9, -LI41 and Lineage I POWV-LB, productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-{beta} (IFN{beta}) and IFN-stimulated genes, with delayed IFN{beta} secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion a subset of POWV infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. ImportanceWe isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculating VeroE6 cells with POWV containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell to cell spread. POWV-LI9, -LI41, and Lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower chamber pericytes, suggesting a mechanism for POWV transmission across BBB. POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB, and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

microbiology↗