Biology subjects

Moffat, J. F.

Publications and source records attributed to Moffat, J. F..

6 recordsLinked to original sources

bioRxiv2026

Fixed Nonsynonymous vOka Mutations in IE62 Drive Varicella-Zoster Virus Attenuation in Human Skin

The live, attenuated vaccine for Varicella-Zoster Virus (VZV) protects against chickenpox in children and adults. However, the vaccine strain Oka (vOka) establishes latency and may reactivate. The molecular basis of vOka attenuation remains unknown. We investigated whether three high-frequency single-nucleotide polymorphisms (SNPs) that differentiate vOka from parental strain Oka (pOka) are responsible: S628G and R958G in the immediate-early regulatory protein IE62, and *130R in the membrane protein ORF0. We evaluated how these fixed SNPs mediate the attenuated growth phenotype of VZV and diminish skin pathology by creating recombinant pOka VZV carrying these SNPs in the wild-type background. These were assessed in cells and human skin organ cultures (SOCs). In epithelial and fibroblast cell lines, the individual IE62 mutations slightly delayed viral growth at 8 - 24 hours post-infection but not in human epidermal keratinocytes (htert-HEK). The fixed ORF62 SNPs also affected ORF62 transcription, IE62 protein abundance at early times, and delayed IE62 accumulation in the cytoplasm later during infection. The *130R mutation in ORF0 conferred a growth advantage in culture, suggesting it is an adaptation to cell culture. While there was donor-to-donor variability and differences in overall virus spread in SOC, the R958G and *130R conferred an attenuated growth phenotype similar to that of vOka in SOC. Histopathologic analysis of VZV-infected skin sections revealed that, similar to vOka, S628G and R958G in IE62 limited the spread of VZV skin lesions. Our findings underscore the critical roles of conserved vOka SNPs in understanding the attenuation of the live vaccine strain vOka.

microbiology↗

bioRxiv2024

Targeting the host transcription factor HSF1 prevents human cytomegalovirus replication in vitro and in vivo

FDA-approved antivirals against HCMV have several limitations, including only targeting the later stages of the viral replication cycle, adverse side effects, and the emergence of drug-resistant strains. Antivirals targeting host factors specifically activated within infected cells and necessary for viral replication could address the current drawbacks of anti-HCMV standard-of-care drugs. In this study, we found HCMV infection stimulated the activation of the stress response transcription factor heat shock transcription factor 1 (HSF1). HCMV entry into fibroblasts rapidly increased HSF1 activity and subsequent relocalization from the cytoplasm to the nucleus, which was maintained throughout viral replication and in contrast to the transient burst of activity induced by canonical heat shock. Prophylactic pharmacological inhibition or genetic depletion of HSF1 prior to HCMV infection attenuated the expression of all classes of viral genes, including immediate early (IE) genes, and virus production, suggesting HSF1 promotes the earliest stages of the viral replication cycle. Therapeutic treatment with SISU-102, an HSF1 inhibitor tool compound, after IE expression also reduced the levels of L proteins and progeny production, suggesting HSF1 regulates multiple steps along the HCMV replication cycle. Leveraging a newly developed human skin xenograft transplant murine model, we found prophylactic treatment with SISU-102 significantly attenuated viral replication in transplanted human skin xenografts as well as viral dissemination to distal sites. These data demonstrate HCMV infection rapidly activates and relocalizes HSF1 to the nucleus to promote viral replication, which can be exploited as a host-directed antiviral strategy. One Sentence SummaryInhibiting of HSF1 as a host-directed antiviral therapy attenuates HCMV replication in vitro and in vivo.

microbiology↗

bioRxiv2022

An Acyclic Phosphonate Prodrug of HPMPC is Effective Against VZV in Skin Organ Culture and Mice

Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC50 of 4 nM and CC50 of 0.20 M, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC.

microbiology↗

bioRxiv2020

β-L-1- uracil (L-BHDU) effectiveness against varicella-zoster virus and herpes simplex virus type 1 depends on thymidine kinase activity

{beta}-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 {micro}M in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TKG22R-GST and TKR130Q- GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDUR virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.

microbiology↗

bioRxiv2020

β-L-1- Uracil (L-BHDU) Inhibits Varicella Zoster Virus Replication by Depleting the Cellular dTTP Pool

{beta}-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) inhibits varicella zoster virus (VZV) replication in cultured cells, human skin, and in SCID-Hu mice with skin xenografts. VZV thymidine kinase converts L-BHDU to monophosphate (MP) and diphosphate (DP) forms, but the triphosphate form was not detected in infected cells and the antiviral mechanism was unknown. Given its similar structure to uridine, we asked if L-BHDU interfered with viral DNA replication via inhibition of the purine and/or pyrimidine biosynthesis pathways. Addition of purines to the medium was unable to restore VZV replication in the presence of L-BHDU. In contrast, excess thymidine and uridine in proportion to L-BHDU restored VZV replication, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. However, addition of thymidine and uridine failed to restore VZV replication in non-dividing cells treated with L-BHDU. Like other herpesviruses, VZV infection increased thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool by nearly 4-fold. The active form(s) of L-BHDU did not interfere with cellular metabolism, suggesting viral target(s).

microbiology↗

bioRxiv2018

Blocking the alternative sigma factor RpoN reduces virulence of Pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

Multidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria. Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment. P. aeruginosa has virulence factors that are regulated by sigma factors in response to the tissue microenvironment. The alternative sigma factor, RpoN ({sigma}54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a \"molecular roadblock\", binding RpoN consensus promoters at the -24 site and blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains both in vitro and in vivo, but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa - C. elegans infection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strain P. aeruginosa PA19660 Xen5. Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

microbiology↗