Biology subjects

Saladino, N.

Publications and source records attributed to Saladino, N..

4 recordsLinked to original sources

bioRxiv2026

Nucleus remodeling activity is conserved amongst diverse SIV Vpr isolates

Human immunodeficiency virus (HIV) encodes four accessory proteins that are essential for virus replication in vivo, primarily through the counteraction of host innate immune defense mechanisms. One of these proteins, Vpr, induces constitutive DNA damage repair (DDR) signaling to drive global epigenetic remodeling and activation of transcription programs that enhance HIV-1 promoter activity during acute infection and virus reactivation from latency. Vpr is conserved amongst diverse simian immunodeficiency virus (SIV) strains; however, the evolutionary breadth of Vpr's nucleus remodeling activity has yet to be thoroughly characterized. Here, we investigate a diverse panel of 16 SIV Vpr isolates and demonstrate that 13 out of 16 are capable of significantly activating DDR signaling compared to control infected cells. Moreover, cells infected with these isolates also exhibit increased abundance of two histone marks associated with transcription and euchromatin formation, as well as increased activation of two transcription factors known to be critical for HIV-1 promoter activity. Furthermore, site-directed mutagenesis of a highly homologous SIV Vpr isolate that failed to engage the DDR response revealed previously uncharacterized amino acid residues required for HIV-1 Vpr DDR engagement. Finally, structural modeling and functional analyses revealed that phylogenetically diverse Vpr isolates from SIV African green monkey strains induce nucleus remodeling through an evolutionarily distinct set of amino acid residues. Together, these findings demonstrate that hijacking of DDR responses to promote remodeling of the nuclear environment is a broadly conserved Vpr function.

microbiology↗

bioRxiv2025

HIV-1 Vpr drives epigenetic remodeling to enhance virus transcription and latency reactivation

Despite decades of research, the primary proviral function of the HIV-1 Vpr accessory protein remains enigmatic. Vpr is essential for pathogenesis in vivo and for virus replication in myeloid cells, but the underlying cause-and-effect mechanism(s) driving these phenomena are poorly understood. Canonically, Vpr hijacks a cellular ubiquitin ligase complex to target several dozen host proteins for proteasomal degradation. Many of these substrates were recently revealed to be involved in DNA damage repair (DDR), which rationalizes the longstanding observation that Vpr induces constitutive activation of DDR signaling. Here, we use a combination of functional, biochemical, and genetic approaches establish a clear mechanistic link between Vpr-induced DDR signaling and remodeling of the epigenetic landscape to enhance HIV-1 promoter activity during acute infection and virus reactivation from latency. Functional, genetic, and bimolecular fluorescence complementation experiments reveal that Vpr utilizes degradation-dependent and -independent mechanisms to induce epigenetic remodeling and that Vpr segregates into two discrete pools with dedicated activities--A multimeric pool in the nucleus that is associated with chromatin and a monomeric pool associated with DCAF1 in the cytoplasm. Vpr function in remodeling the nuclear environment is present in common HIV-1 subtypes worldwide and provides a mechanistic rationale for its essentiality in virus replication. Author summaryWhile HIV-1 Vpr plays an essential role in virus replication, the molecular mechanisms underlying its essentiality remain enigmatic. Vprs best characterized function is the ability to induce the depletion of several dozen host proteins by hijacking a cellular E3-ubiquitin ligase complex. Here, we establish that Vpr promotes global epigenetic remodeling to enhance HIV-1 promoter activity during acute infection and virus reactivation from latency. We demonstrate that epigenetic remodeling activity is linked to Vprs ability to induce constitutive DNA damage repair signaling, and that it occurs through both degradation-dependent and -independent mechanisms. Moreover, this Vpr function is present in common HIV-1 subtypes circulating globally. This study provides novel mechanistic insights into how HIV-1 exploits host DNA repair pathways and sheds light on Vprs proviral function.

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↗