Biology subjects

Himmler, G. E.

Publications and source records attributed to Himmler, G. E..

4 recordsLinked to original sources

bioRxiv2026

Powassan Virus LB Neurovirulence and Lethality is Determined by Envelope Protein Domain III Residues

Powassan Viruses (POWV) are tick-borne Flaviviruses that cause lethal encephalitis and long-term neurologic sequelae. POWVs are divided into two lineages that share 86% genetic identity. POWV-LB (Lin I) was isolated from a fatal case of encephalitis, while POWV-LI9 (Lin II) is a tick-derived isolate that causes age-dependent lethality in mice. Domain III of Flavivirus envelope proteins (EDIII) directs cell attachment, and determines murine neuropathology and lethality. Here we developed POWV LB reverse genetics, analyzed the CNS of LB and recLB mutant infected mice, and assessed the role of EDIII residues in LB pathogenesis. LB infection of 50-week-old B6 mice focally activated microglia in the midbrain and cerebellum discrete from, and independent of, the location of envelope protein in the cerebral cortex, hippocampus and thalamus. Reverse genetic analysis of LB EDIII residues revealed that mutating D308N only partially reduced LB neuropathology and lethality. However, mutating D308N along with an adjacent EDIII residue, A310T, completely abolished LB-D308N/A310T neurovirulence, neuroinvasion and lethality, yet elicited neutralizing antibody responses that protected mice from a lethal WT LB challenge. Signifying changes in neurotropism, LB-D308N/A310T failed to activate microglia or yield detectable POWV RNA and antigen in the CNS. Overall, recombinant LB-D308N/A310T mutants fail to induce neurologic symptoms or neuropathology while eliciting a protective immune response. These findings reveal that EDIII residues determine the neurovirulence and lethality of lineage I POWVs and that genetically modifying EDIII provides a mechanism for POWV attenuation and vaccine development. ImportancePowassan virus causes lethal encephalitis and long-term neurological sequalae in survivors; however, there are currently no licensed POWV vaccines or therapeutics. POWVs are comprised of two genetic lineages (LB-Lin I; LI9 Lin II) that are derived from discrete ticks and small mammal hosts. Flavivirus envelope proteins determine cell attachment, viral tropism, and neurovirulent spread and lethality in mice. Here we created a POWV LB reverse genetics system and assessed mutations that determine LB neurovirulence in 50 wk old mice. Studies reveal novel LB neuropathology with divergent patterns of gliosis and POWV antigen expression within the CNS. Mice infected with an LB-D308N/A310T envelope mutant elicited responses that protect mice from a lethal LB challenge and failed to enter the CNS, cause neurologic symptoms, CNS pathology or lethality. LB reverse genetics permits defining determinants of LB neurovirulence that are required for rational vaccine development.

microbiology↗

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↗

bioRxiv2023

Immune response modulation by Pseudomonas aeruginosa persister cells

Bacterial persister cells - a metabolically dormant subpopulation tolerant to antimicrobials - contribute to chronic infections and are thought to evade host immunity. In this work, we studied the ability of Pseudomonas aeruginosa persister cells to withstand host innate immunity. We found that persister cells resist MAC-mediated killing by the complement system despite being bound by complement protein C3b at levels similar to regular vegetative cells, in part due to reduced bound C5b - and are engulfed at a lower rate (10-100 fold), even following opsonization. Once engulfed, persister cells resist killing and, contrary to regular vegetative cells which induce a M1 favored (CD80+/CD86+/CD206-, high levels of CXCL-8, IL-6, and TNF-) macrophage polarization, they initially induce a M2 favored macrophage polarization (CD80+/CD86+/CD206+, high levels of IL-10, and intermediate levels of CXCL-8, IL-6, and TNF-), which is skewed towards M1 favored polarization (high levels of CXCL-8 and IL-6, lower levels of IL-10) by 24 hours of infection, once persister cells awaken. Overall, our findings further establish the ability of persister cells to evade the innate host response and to contribute chronic infections.

microbiology↗