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Department of Microbiology
Tufts University, 1985, Ph.D.
612-624-9933 - office
612-626-0623 - fax
Virus-host interactions, viral entry, translational control in virus-infected cells, viral immune avoidance mechanisms
Diagram of the reovirus life cycle
Mammalian orthoreoviruses are prototypical members of the family Reoviridae, which includes the pathogenic rotaviruses, coltiviruses and orbiviruses. Orthoreoviruses (reoviruses) are poorly pathogenic in humans. This characteristic, together with their preferential replication in transformed cells, has led to their recent development as oncolytic agents for the treatment of human tumors. However, despite the ongoing trials of reovirus therapy in humans, relatively little is known about the molecular determinants (viral and cellular) that regulate infection. My laboratory uses mammalian reoviruses to understand molecular interactions that influence the outcome of viral infection. Our studies have focused on functions of sigma 3, one of the most abundant proteins in the reovirus virion (see cryo-EM diagram below) and reovirus-infected cell. Sigma 3 functions in two distinct, yet fundamentally important steps in the viral life cycle: penetration of the viral particle into the cell cytoplasm and the regulation of protein synthesis/interferon-resistance in the infected cell.
Surface-shaded views of reovirus TIL (left) showing a split particle composed of a TIL viron and an ISVP and (right) a split particle composed of a T1L ISVP and a core particle. Pictures are based on three-dimensional reconstruction of data obtained by cryoelectron microscopy. Image provided by Dr. Max Nibert, Harvard Medical School (my favorite collaborator!)
Space filling diagram of sigma 3 showing conserved and non-conserved residues. Residues in red are not conserved among different serotypes and isolates. Conserved residues are in gray.Views (A) and (B) are rotated by 180 degrees with respect to each other about the long axis. Residues thought, based on mutational analysis, to be involved in interactions with mu 1 are shown in blue. Lys 293, thought to be involved in the binding of dsRNA, is shown in yellow. The protease-hypersensitive loop is at the bottom right of (A) and at the top right of (B). From; Olland, A.M., Jane-Valbuena, J., Schiff, L.A., Nibert, M.L., and Harrison, S.C. (2001) Structure of the reovirus outer capsid protein sigma 3 at 1.8 angstroms resolution. EMBO J. 20(5):1-11.
Search Entrez Browser for Schiff L
Unlike other enteric viruses that typically require extracellular proteolysis to initiate infection, reoviruses appear to exploit both intracellular and extracellular enzymes to degrade sigma 3 and prepare the particle for viral entry. Our recent work has focused on how proteolysis influences the capacity of reovirus to infect cells and cause disease. Joe Golden discovered that the requirement for sigma 3 removal represents a significant host-range determinant in reovirus infection. He found that many cells that otherwise support efficient reovirus replication, restrict infection at this step in the lifecycle. Joe’s thesis work went on to demonstrate that reovirus strains differ in their susceptibility to uncoating by specific proteases and that this can impact cellular host range. This work ultimately contributed to the important finding that reoviruses replicate preferentially in tumor cells because they are more efficiently uncoated in cultured tumor cells and the in vivo tumor microenvironment. Joe’s work also showed that distinct proteases mediate uncoating in different cell types. Studies in cells that express the cysteine protease cathepsin S or the inflammatory serine protease elastase support a model in which the pH-sensitivity of reovirus infection reflects the acid-dependence of particular proteases involved in uncoating, rather than an requisite inherently acid-sensitive step in viral entry.
Our present goals are to identify cellular proteases and viral determinants of proteolysis that regulate reovirus cell entry and to understand how these factors impact infection and pathogenesis in the host. We are using genetic and biochemical approaches to identify sequences within capsid protein sigma 3 that regulate cell entry. We are selecting and characterizing viral mutants with expanded protease sensitivity and we will use infectious particles recoated with recombinant sigma 3 and a newly developed reverse genetic system to identify determinant of protease susceptibility crucial for cell entry. Within the next year, we plan to move our work into animals, using well-developed mouse infection models and knockout mice to investigate the role that specific intracellular and extracellular proteases play in regulating reovirus tropism, spread and disease. Our studies of the molecular determinants of reovirus cell entry will provide fundamental information concerning the biology of non-enveloped viruses and will facilitate the safe and effective use of reoviruses as oncolytic agents to treat human tumors.
The second line of research in my laboratory addresses the fundamental question of how viruses modulate the cellular translation machinery to favor their own replication. We are investigating both the mechanisms by which reovirus shuts off host cell translation and the mechanisms that enable viruses to replicate in the face of global host shutoff. Based on a genetic polymorphism between two prototypic reovirus strains, it has long been hypothesized that the interferon-induced eIF2 alpha kinase, PKR, is responsible for reovirus-induced host shutoff.
However Jen Smith’s thesis work revealed that, while PKR may be entirely responsible for host shutoff after infection with the prototypic strain Jones, other molecules contribute to this phenotype in cells infected with most other reovirus strains. Her infection studies in knockout cells revealed that RNase L, another interferon-induced antiviral molecule, contributes to reovirus-induced host shutoff.
Total profile of 35S-methionine-labeled proteins in reovirus-infected (D,87,J) or uninfected (mock or M) L929 cells. The positions of viral proteins are indicated at the right of each figure. Notice that host protein synthesis is dramatically inhibited after infection with reovirus clone 87 or Jones (J). Host translational shutoff is minimal after infection with strain Dearing (D). The difference in the host translation profiles between the Jones infection and the Dearing infection is determined by the gene encoding the viral protein sigma 3.
Interestingly, while these molecules inhibit host protein synthesis in infected cells, they do not significantly restrict viral replication. In fact, virus yields can be higher in the presence of these ‘antiviral’ molecules! To gain further insight into the causes and consequences of reovirus-induced host shutoff and to better understand how reovirus counters these cellular defenses, we performed a comparative microarray analysis with our colleague Paul Bohjanen (manuscript in preparation). We found that the cellular responses to infection vary quite dramatically depending upon the infecting strain. Interestingly, infection with virus strains that induce host shutoff alters the expression of genes involved in the cellular integrated stress response. These include PERK, an ER resident PKR-like eIF2 alpha kinase and P58IPK, a cellular inhibitor of PKR and PERK. An important goal in the near term is to understand how reovirus infection activates cellular stress pathways. Another important goal is to understand the mechanisms that enable reovirus mRNAs to be efficiently translated in cells in which PERK and PKR phosphorylate and inactivate the eukaryotic translation initiation factor eIF2 alpha . One hypothesis, emanating from the work of Stephen Schmechel in the lab, is that sigma 3 locally inhibits dsRNA-activated molecules such as PKR and RNAse L, to spare translation in areas of viral replication (see figure below). Over the last thirty years, significant insight into translational regulation has been gained from studies of virus-infected cells. Our studies in reovirus-infected cells are likely to provide new and important information regarding mechanisms of eukaroytic translation initiation under conditions of cellular stress.
Current Ph.D. Trainees
Rachel Nygaard (on the left)
Past Ph.D. Trainees:
Jennifer Smith, Ph.D.
Currently a postdoctoral fellow with P. Howley, Harvard Medical School
Joseph Golden, Ph.D.
Currently a postdoctoral fellow at USAMARIID
Stephen Schmechel, M.D/Ph.D.
Currently an Assistant Professor, Department of Laboratory Medicine and Pathology, University of Minnesota
Former undergraduates who have pursued directed research in the lab:
Ross Kedl, Ph.D. (Asst. Professor, Dept. of Immunology, UCHSC), Brent Huberty (Manager, Pharmacia/Upjohn Sterility Testing Laboratory), Pamela Skinner, Ph.D. (Asst. Professor, University of Minnesota Veterinary School), Robert Anderson, M.D./Ph.D. (ASM undergraduate research fellowship awardee), Michael Chute (Manager, BSL3 Laboratory, Biological Defense Research Directorate, Naval Medical Research Center), Jeffrey Ehnstrom, M.D., Kara Thoemke, Ph.D. (Asst. Professor, College of St. Scholastica), Jay Santos (ASM undergraduate research fellowship awardee), Ashley Fuller, MD (OB/Gyn resident, UT-Southwestern), Patrick Laitala (Advanced Microbial Systems), Tseganesh Selameab, M.D. (resident, Boston Medical Center), Amanda Kostyk, Ph.D. (M.D./Ph.D. University of Colorado), Jessica Linke (National Conference on Undergraduate Research participant; Biology teacher, Delano High School), Eva Chung (Mentored through the Presidents Distinguished Faculty Mentor Program, Ph.D. student in Immunology at Duke), Daniel Westholm (Ph.D. candidate, University of Minnesota, Duluth), Brian Finstead (Beckman-Coulter, Production Scientist), Taj Melson (medical student at University of Michigan), Linse Lahti (Junior Scientist University of Minnesota)
Jessie Bahe (currently a high school science teacher)
Linse Lahti (Junior Scientist and Superscuba girl)
Current Teaching at the Undergraduate Level
Recent Teaching at the Graduate Level
Presenter at teaching enhancement workshops sponsored by the University of Minnesota Center for Teaching and Learning Services and the Center for Writing:
Less Yields More: Using Short Writings in Writing Intensive Courses, Winter 2000 and Fall 2001; Using Technology to Enhance Learning and Teaching Objectives, Spring 2001; Enhancing Large Lecture Courses: A Showcase, Fall 2001; Enhancing Large Lecture Courses: Continuing the Discussion, Fall 2001; Making Use of Short and Informal Writing Assignments, Fall 2002, Fall 2003, and Fall 2004; Teaching Writing One-on-One, Fall 2003; Grading Writing, Fall, 2003; Writing-to-Learn, Fall 2003: Teaching with Writing Online, Fall, 2005; Designing writing assignments that promote learning, Fall 2005; Incorporating writing instruction into “content” courses, Spring 2006; Teaching with Writing Online and Grammar matters, Fall 2006; What’s Grammar Got To Do With It, Spring 2007.
Huey (yellow australian shepherd), Jose (long-haired german shepherd) and Steve (herpesvirologist and co-inhabitant of lab space in 1425 Mayo)
Noah Hai Lam Rice (doing some reading in his stroller while visiting the lab
Noah Hai Lam Rice (5 years old, kidratee)
Noah and friend Nikki, Nature of Life peer mentors, 2022