About

Dennis T Brown is the William Neal Reynolds Professor at NC State University, specializing in the structure, function, and assembly of the model membrane containing the Sindbis virus. His laboratory investigates the complex structure of alphaviruses, which are transmitted by blood-sucking insects and composed of multiple proteins forming two icosahedral shells with an intervening membrane bilayer. His research aims to determine the high-resolution structure of the virus using Electron Cryo-Microscopy, complemented by biophysical and biochemical analyses to verify morphological data. Brown's work includes elucidating the pathway of virus component folding and assembly within infected cells, focusing on the disulfide bridging of membrane glycoproteins and their integration into the mature virion. His approach combines molecular and cellular biology techniques, molecular cloning, and site-directed mutagenesis to understand virus assembly processes.

Research topics

• Virology
• Biology
• Microbiology
• Crystallography
• Nanotechnology
• Chemistry
• Condensed matter physics
• Physics
• Materials science
• Biochemistry

Selected publications

• Oxidative stress induced by menadione (vitamin K3) results in non-canonical AQP2 membrane traffcking in LLC-AQP2 cells

  Physiology · 2024-05-01

  articleSenior author

  Oxidative stress affects a wide variety of cellular processes including morphology, protein traffcking, and cell death. Several studies show that reactive oxygen species (ROS) are able to pass through some water channels, and that ROS may modulate aquaporin expression and traffcking. However, a relationship between ROS status and the traffcking of aquaporin-2 (AQP2) has yet to be well-defined. To address this question, we first treated LLC-PK1 cells expressing cmyc-tagged AQP2 (LLC-AQP2 cells) for 30 min with menadione (MK3, 20 μM), an agent widely used to generate ROS in cell culture. Similar to vasopressin (VP), MK3 induced significant AQP2 accumulation in the plasma membrane of these cells. Using LLC-AQP2 cells expressing secreted soluble green fluorescent protein (ss-GFP), we revealed significantly increased vesicle exocytosis upon treatment with MK3. Surprisingly, MK3 did not affect rhodamine-labeled dextran endocytosis, suggesting that the MK3 effect is mainly due an increase in AQP2 exocytosis into the plasma membrane. Next, we studied the role of AQP2 c-terminal serine 256 phosphorylation in this process. MK3 treatment resulted in significant plasma membrane accumulation of the non-phosphorylatable AQP2 S256A mutant, whereas VP had no effect, as previously reported. To understand the contribution of protein kinase A (PKA) to MK3-mediated AQP2 traffcking, we used CRISPR-generated LLC-AQP2,koPKA cells that lack PKA isoforms. Interestingly, an increase in AQP2 membrane accumulation was still observed, suggesting that the MK3 effect is PKA-independent. By western blotting with anti-phospho-AQP2 antibodies, we found that MK3 did not affect phosphorylation of the critical S256 residue, but in contrast to VP, MK3 actually increased the phosphorylation level of S261 instead of reducing it. Finally, we showed that AQP2 accumulation in both LLC-AQP2 and LLC-AQP2 S256A cells was inhibited in the presence of glutathione (5 mM), an anti-oxidant or reducing agent. Interestingly, western blot analysis showed that glutathione abolished the MK3-induced increase of S261 AQP2 phosphorylation. In summary, our study shows that menadione (MK3) induces AQP2 membrane accumulation in LLC-AQP2 cells, suggesting a role of ROS in this process. This effect is largely due a change in AQP2 vesicle exocytosis. It does not require phosphorylation of the c-terminal S256 residue of AQP2, but MK3 does cause increased S261 phosphorylation. While incompletely understood at present, these data reveal that alternative, probably ROS-related, regulatory mechanisms can be involved in AQP2 traffcking and recycling. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

  PublisherDOI

• Tizoxanide Antiviral Activity on Dengue Virus Replication

  Viruses · 2023 · 7 citations

  • Virology
  • Biology
  • Microbiology

  Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.

  PublisherOA PDFDOI

• Doping dependent electronic and magnetic ordering in mixed-valent La_1−xSr_xMnO₃ thin films

  Materials Research Express · 2021 · 1 citations

  • Condensed matter physics
  • Materials science
  • Crystallography

  Abstract We have investigated the collective electronic and magnetic orderings of a series of La 1− x Sr x MnO 3 thin films grown epitaxially strained to (001) oriented strontium titanate substrates as a function of doping, x , for 0 ≤ x ≤ 0.4. We find that the ground states of these crystalline thin films are, in general, consistent with that observed in bulk crystals and thin film samples synthesized under a multitude of techniques. Our systematic study, however, reveal subtle features in the temperature dependent electronic transport and magnetization measurements, which presumably arise due to Jahn-Teller type distortions in the lattice for particular doping levels. For the parent compound LaMnO 3 ( x = 0), we report evidence of a strain-induced ferromagnetic ordering in contrast to the antiferromagnetic ground state found in bulk crystals.

  PublisherDOI

• Quantitative proteomic analysis of the tizoxanide effect in vero cells

  Scientific Reports · 2020 · 7 citations

  • Virology
  • Biology
  • Microbiology

  Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.

  PublisherOA PDFDOI

• Role of the vacuolar ATPase in the Alphavirus replication cycle

  Heliyon · 2018-07-01 · 8 citations

  articleOpen accessSenior author

  infection.

  PublisherOA PDFDOI

• The ammonia transporter RhCG modulates urinary acidification by interacting with the vacuolar proton-ATPases in renal intercalated cells

  Kidney International · 2017-10-18 · 18 citations

  articleOpen access
  PublisherOA PDFDOI

• Characterization and Correction of Olfactory Deficits in Kidney Disease

  Journal of the American Society of Nephrology · 2017-08-03 · 41 citations

  articleOpen accessCorresponding

  Patients with CKD suffer from food aversion, anorexia, and malnutrition. Although olfaction has a significant role in determining food flavor, our understanding of olfactory impairment and of the olfaction-nutrition axis in patients with kidney disease is limited. We quantified odor identification, odor threshold, and subjective odor perception in a cohort ( n =161) comprising 36 participants with CKD, 100 participants with ESRD, and 25 controls. We investigated olfaction-nutrition associations in these participants and examined a novel intervention to improve olfaction in ESRD. The mean odor identification score was lower in patients with CKD (75.6%±13.1%; P =0.02) and ESRD (66.8%±15.1%; P <0.001) than in controls (83.6%±11.4%). Patients with ESRD exhibited higher odor threshold than the remaining participants exhibited. All groups had similar scores for subjective smell assessment. In multivariable adjusted analyses, kidney disease associated with increased odds of odor identification deficits (odds ratio, 4.80; 95% confidence interval, 1.94 to 11.89). A reduction in odor identification score was associated with higher subjective global assessment score and lower serum total cholesterol, LDL cholesterol, and albumin concentrations. We found no associations between odor threshold and nutritional parameters. In a proof of concept, 6-week, open-label clinical trial, intranasal theophylline (an epithelial membrane transport and proton secretion activator) increased odor identification score in five out of seven (71%) patients with ESRD. In conclusion, patients with kidney disease have olfactory deficits that may influence their nutritional status. Our preliminary results regarding olfactory improvement using intranasal theophylline warrant confirmation in a randomized controlled trial.

  PublisherOA PDFDOI

• Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly

  Journal of Virology · 2016-07-14 · 1 citations

  articleOpen accessSenior author

  UNLABELLED: A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface. (35)S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer. IMPORTANCE: Our data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.

  PublisherOA PDFDOI

• Locations of Carbohydrate Sites on Alphavirus Glycoproteins Show that E1 Forms an Icosahedral Scaffold

  WORLD SCIENTIFIC eBooks · 2014-05-01 · 1 citations

  book-chapter
  PublisherDOI

• Structural Differences Observed in Arboviruses of the Alphavirus and Flavivirus Genera

  Advances in Virology · 2014-01-01 · 28 citations

  reviewOpen access

  Arthropod borne viruses have developed a complex life cycle adapted to alternate between insect and vertebrate hosts. These arthropod-borne viruses belong mainly to the families Togaviridae, Flaviviridae, and Bunyaviridae. This group of viruses contains many pathogens that cause febrile, hemorrhagic, and encephalitic disease or arthritic symptoms which can be persistent. It has been appreciated for many years that these viruses were evolutionarily adapted to function in the highly divergent cellular environments of both insect and mammalian phyla. These viruses are hybrid in nature, containing viral-encoded RNA and proteins which are glycosylated by the host and encapsulate viral nucleocapsids in the context of a host-derived membrane. From a structural perspective, these virus particles are macromolecular machines adapted in design to assemble into a packaging and delivery system for the virus genome and, only when associated with the conditions appropriate for a productive infection, to disassemble and deliver the RNA cargo. It was initially assumed that the structures of the virus from both hosts were equivalent. New evidence that alphaviruses and flaviviruses can exist in more than one conformation postenvelopment will be discussed in this review. The data are limited but should refocus the field of structural biology on the metastable nature of these viruses.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01AI014710

  NIH · $991k · 1997

• NIH Grant R01DK042956

  NIH · $4.3M · 2008

• NIH Grant R37DK042956

  NIH · $4.0M · 2018

• Human Genetics and Microbiome Core

  NIH · $38.3M · 1997–2027

• NIH Grant R22AI019545

  NIH · $88k · 1986

Frequent coauthors

• Raquel Hernandez

  42 shared

• Sylvie Breton

  Massachusetts General Hospital

  21 shared

• Ivan Sabolić

  University Hospital Centre Zagreb

  17 shared

• Davis Ferreira

  Duke University Hospital

  16 shared

• Carol M. Herak–Kramberger

  Institute for Medical Research and Occupational Health

  14 shared

• Winnie Shum

  Center for Excellence in Molecular Cell Science

  13 shared

• Jean‐Marc Verbavatz

  Université Paris Cité

  12 shared

• Dennis A. Ausiello

  Center for Assessment

  12 shared

Labs

• Molecular and Structural BiochemistryPI

Education

• Ph.D, Molecular Biology

  University of Pennsylvania

  1967

• BA, Biology/Chemistry

  University of Pennsylvania

  1964