The Department of Microbiology has strength in many different areas of viral research. Our faculty perform research on a wide spectrum of fundamental virological problems including: 1) the analysis of viral evolution; 2) the mechanisms of viral genome replication and the function of both cellular and viral proteins in this process; 3) the mechanisms of viral genome encapsidation and assembly; 4) understanding virus-host interactions involved in inhibiting the cellular antiviral response; 5) the mechanisms of viral control of cellulartranscription and translation; 5) understanding the differences in the adaptive immune response during acute and persistent viral infections. Our unique combination of structural and molecular virology research results not only in the generation of basic knowledge about fundamental aspects of virus replication and pathogenesis on the molecular and organismal levels, but also provides a strong foundation for applied research. Our faculty perform a wide variety of studies aimed at the development of new drugs capable of interfering with different processes in virus replication and assembly, the design new viral vectors for the expression of heterologous genetic information and drug delivery, and the design of new efficient and highly attenuated vaccine candidates.
Terje Dokland, Ph.D.
Structure and assembly of viruses and viral proteins
The research in my lab is focused on the structures of viruses and viral proteins, and the macromolecular assembly processes that gives rise to functional virions. One project is on the role of “helper” bacteriophages in the mobilization of pathogenicity islands (SaPIs) in Staphylococcus aureus. SaPIs lack genes encoding structural gene products, but are packaged into viral particles using proteins encoded by the helper. In many cases the capsids formed are smaller than those normally assembled by the virus itself. We are interested in the regulation of this size determination process, which we study with a combination of structural and biochemical techniques, including cryo-EM and X-ray crystallography. Other projects focus on the bacteriophage P2/P4 system and on the agricultural pathogen PRRSV, an RNA virus belonging to the arterivirus family that infects pigs.
Ilya Frolov, Ph.D.
My research is focused on dissecting the molecular mechanisms of alphavirus replication and interaction with host cells. In this direction, we are studying the RNA promoter elements and the mechanism of formation of alphavirus replication complexes, define functions and interactions of their cellular and viral components. Another direction of our research is aimed at understanding the mechanism of Venezuelan equine encephalitis virus particle assembly and RNA encapsidation. These data are being used for development of new vaccine candidates against alphavirus infections.
Elena Frolova, Ph.D.
Our research interests are geared towards expanding our understanding of alphavirus pathogenesis at the molecular and cellular levels. Alphaviruses are circulating in the Central, South and North Americas and cause periodic, extensive equine epizootics and epidemics of encephalitis with frequent lethal outcomes and neurological sequelae in humans. The main objective of out lab is to investigate the functions of virus-specific nonstructural and structural proteins in virus replication and inhibition of the cellular antiviral response. Our studies have demonstrated that geographically isolated New World alphaviruses, such as Venezuelan equine encephalitis virus and Old World alphaviruses, such as Sindbis and chikungunya viruses, have developed fundamental differences in their mechanisms for downregulating cellular transcription and translation, which represent the main means of alphavirus inactivation of antiviral genes. The detailed characterization of these mechanisms provides a strong basis for the rational design of recombinant alphaviruses with programmed, irreversibly attenuated, cell-restricted phenotypes. These genetically modified aphaviruses can, subsequently, be safely used for vaccine development. Other projects in the lab are aimed at the production and purification of alphavirus nonstructural proteins, characterization of their enzymatic activities and design of assays for screening small molecule libraries in order to identify new antiviral drugs.
Todd Green, Ph.D.
Dr. Green’s lab uses structural techniques to study proteins from negative-stranded RNA viruses that are involved in polynucleotide synthesis. In order to gain a better understanding of the replication cycle of this class of viruses, my lab is studying the molecular structure of vesicular stomatitis virus (VSV), a prototype of this group. Using state-of-the-art techniques, such as x-ray crystallography and cryo-electron microscopy, we have observed atomic-level snapshots of how the viral capsid protein encapsidates and protects the genomic RNA, how this template is accessed by the viral polymerase co-factors, and how the capsid structure is affected by specific RNA sequences. Moving forward, we continue to investigate the mechanisms of viral transcription and replication by structural and biochemical techniques. Better understanding of these processes will yield new avenues for future therapeutic design against this group of viruses.
Mengxi Jiang, Ph.D.
My laboratory studies how viral pathogens interface with the host. As obligate intracellular parasites, viruses often hijack or inactivate cellular processes to benefit their own replication at various stages of their life cycle. We are particularly interested in the roles played by cellular nuclear components during DNA virus infections. Our work has focused on a human pathogen, BK polyomavirus (BKPyV), a small DNA tumor virus that causes severe disease in kidney and bone marrow transplant patients. In kidney transplant recipients, BKPyV reactivation leads to the development of polyomavirus-associated nephropathy (PVAN), a serious complication that can result in graft rejection. The major areas of research in the laboratory are: 1) dissect the relationships between BKPyV and the cellular DNA damage response; 2) determine molecular triggers that lead to BKPyV reactivation in transplant patients; 3) identify novel nuclear pathways and components that contribute to BKPyV replication. We use a combination of state-of-the-art genetic, molecular and cell biology, and proteomic techniques to address these questions. The long-term goal of this research is to define host-pathogen interactions that impact the outcomes of polyomavirus infection and pathogenesis.
Elliot Lefkowitz, Ph.D.
My research interests are directed at contributing to the understanding of microbial genomics and evolution by developing and utilizing computational tools and bioinformatics techniques to mine sequence and other data for significant patterns characteristic of function and/or evolution. This work has included the development of new algorithms for the detection of viral regulatory motifs; tools for the identification of viral genes; the development and utilization of High Performance and Grid Computing tools for bioinformatics analysis; and the development and use of tools for analyzing patterns of viral evolution. I have also been involved in the development of databases, web applications, and analysis tools for the sequencing and annotation of several complete bacterial and viral genomes. This includes development of the Viral Bioinformatics Resource Center, one of the original NIH-sponsored Bioinformatics Resource Centers (BRCs) for Biodefense and Emerging or Re-Emerging Infectious Diseases. My work includes research on the genomics and evolutionary history of large DNA viruses as well as several different RNA virus species including Human papillomavirus, Hepatitis C virus and Dengue virus.
Ongoing research projects in my group are focused on the analysis of, and the development of tools to support the analysis of the genomics and evolution of the large DNA viruses in the family Poxviridae. Poxviruses are highly successful pathogens, known to infect a variety of hosts. The family Poxviridae includes variola virus, the causative agent of smallpox, which has been eradicated as a public health threat but could potentially reemerge as a bioterrorist threat. The risk scenario includes other animal poxviruses as well as genetically engineered or synthetically derived poxviruses. Past research on orthologous viral gene sets has defined some of the evolutionary relationships between members of the Poxviridae family. But it has not been clear how variation between family members arose in the past—an important issue in understanding how these viruses may vary and possibly produce future threats. Therefore the goal of our research has been to better understand the viral genotypic-phenotypic relationships that result in wide differences in host range and pathogenicity, as well as the evolutionary mechanisms involved in genome variation. Our work has included a comprehensive analysis of all proteins encoded by viruses of the family Poxviridae, to assess the evolutionary history of genes likely acquired through horizontal gene transfer. This effort has allowed us to predict and date the patterns of host gene acquisition throughout the evolutionary history of this virus family, and helps us to better understand the molecular mechanisms of survival and pathogenesis employed by viruses of this family at different stages of their evolution.Frances Lund, Ph.D.
One of the major research objectives in the Lund laboratory is to identify the key factors that regulate the balance between protective and pathogenic immune responses to viral infection. In one project we use the influenza infection model to evaluate how B cells and the antibodies made by B cells contribute to local pulmonary protection from reinfections with the same or different strains of the influenza virus. We use genetically modified strains of mice as well as altered influenza viruses to identify the key pathogen and immune system-derived signals that initiate the early innate as well as later adaptive B cell response to flu. We then use this information to evaluate B cell responses in humans following natural infection and vaccination with the long-term goal of designing more protective influenza vaccines. In a second project, we evaluate immune-mediated pulmonary pathology in animals infected with influenza virus. In particular, we focus on the early innate immune response and evaluate how chemokines modulate pulmonary inflammation and tissue damage. The goal of this research is to identify therapeutics that dampen the damaging lung inflammation that often accompanies infection with the most pathogenic strains of flu.
Guangxiang G Luo, M.D.
Our overall objective is to combine reverse genetic, biochemical, cell biological, proteomic, and transgenic approaches toward a thorough understanding of the molecular mechanisms of hepatitis C virus (HCV) infection, replication, virion assembly, virus-host interaction, and molecular pathogenesis. Specifically, our research programs include: 1) defining the roles of viral and cellular proteins in HCV infection, replication, and assembly; 2) developing small animal models of HCV infection, replication, pathogenesis, and carcinogenesis; 3) illustrating virus-host interactions in vitro and in vivo; 4) understanding the molecular bases underlying the HCV-induced carcinogenesis and profiling hepatic cancer stem cells; 5) identifying novel targets and antiviral drugs for treatment of hepatitis C; 6) developing effective HCV vaccines; and 7) determining the underlying molecular mechanism of HCV and HIV interaction in cell culture and in vivo. Additionally, we are interested in molecular genetic analysis of other RNA viruses, including hepatitis E virus, West-Nile virus, Dengue Virus, and respiratory viruses such as influenza viruses and respiratory syncytial virus.
Jan Novak, Ph.D.
Dr. Novak’s research interests in Virology include studies of glycosylation of viral glycoproteins and the effect of differential glycosylation on infectivity and immunogenicity. For example, understanding the role of differential glycosylation of HIV-1 envelope glycoprotein on its multiple functions will impact development of new effective HIV vaccines.
Peter Prevelige, Ph.D.
Virus Structure and Assembly
My laboratory uses a variety of biophysical and structural approaches to approach fundament questions in virus structure and assembly. We are particularly interested in questions of form determination, for how icosahedral viruses assemble from subunits which high fidelity, or why some viral capsid such as HIV adopt different morphologies. Among the techniques that we utilize are: cryo-EM, NMR, mass spectrometry, single molecule spectroscopy, light scattering, and kinetic analysis.
Virus Based Bio-Nanotechnology
Virus particles can be viewed as nanoscale structural scaffolds precisely defined down to the atomic level. Well understood techniques of protein chemistry and molecular biology provide facile tools for the selective modification of individual amino acids. These modified viral particles can be used to display targeting ligands for biomedical imaging and delivery (theranostics) applications or alternatively as a route to well-defined nanoscale materials. Our virus based bio-nano projects utilize our detailed knowledge of the structure and assembly of the 60nm capsid of the bacteriophage P22 to address both of these classes of applications.
Jamil S. Saad, Ph.D.
HIV-1 replication is strongly dependent on the cellular machinery to produce progeny virus. The discovery of cellular factors that participate in HIV replication pathways has provided new insights into the molecular basis of virus–host cell interactions. Elucidation of the molecular interactions between the host cell and HIV are important for understanding the virus replication and the subsequent cytopathogenesis in the infected cell, which will aid in the development of more efficient antiviral drugs. We are interested in the underlying structural basis by which HIV proteins interact with cellular constituents during the virus replication cycle. A major component of our research program is directed towards understanding key protein-protein and protein-membrane interactions that are critical for HIV replication. Another aspect of our research is to identify small molecule inhibitors that are able to block virus-host protein-protein complexes and ultimately serve as potential anti HIV drugs. The results generated from this research will provide new insights into these mechanisms and identify new attractive targets that will ultimately aid in rational drug design.
Hubert Tse, Ph.D.
An overarching theme in the Tse laboratory is to determine the involvement of oxidative stress and immune-mediated effector mechanisms associated with pancreatic beta-cell destruction in Type 1 diabetes (T1D). Currently, no cure exists for this polygenic autoimmune disease and worldwide incidence is steadily climbing. Changes in population genetics over generations cannot account for the sudden rise in T1D incidence. It has been suggested that a “viral trigger” can initiate T1D, but the exact mechanism(s) is unknown. Epidemiological data with newly diagnosed T1D patients has demonstrated an increase in enteroviral RNA and antibodies against diabetogenic enteroviruses including Coxsackie B4 (CB4) in the blood as compared to non-diabetics. In T1D, anti-viral responses to diabetogenic CB4 and Encephalomyocarditis viruses (EMCV) are highly understudied, but more importantly, the role of oxidative stress and reactive oxygen species (ROS) to influence innate immunity is not well defined. The importance of ROS synthesis in T1D has recently been demonstrated by our laboratory, a dominant negative p47phox (Ncf1m1J) mutation of the NADPH oxidase complex was introgressed into the non-obese diabetic (NOD) mouse, a murine model for studying T1D. NOD.Ncf1m1J mice are impaired in ROS synthesis and highly resistant to spontaneous diabetes and adoptive transfer of diabetes with diabetogenic T cells. T1D-resistance in NOD.Ncf1m1J mice may be partly explained by dampened innate immune responses after viral infection. The decrease in ROS and pro-inflammatory cytokine synthesis observed by EMCV-infected NOD.Ncf1m1J macrophages may directly prevent pancreatic beta-cell destruction and/or inhibit bystander activation of autoreactive CD4 and CD8 T cells. Currently, the synergy of oxidative stress on the activation of innate immune responses to diabetogenic viral triggers (CB4, EMCV) and autoreactive T cells in Type 1 diabetes is being defined.
Mark R. Walter, Ph.D.
Structural biology of viral proteins that disrupt cytokine signaling: An ongoing goal of the laboratory is to design molecules that enhance or prevent cytokine signaling. One way to understand how to manipulate cytokine signals is to understand the mechanisms used by viruses to accomplish this task. Pox and herpes viruses have designed novel proteins to disrupt cytokine signaling upon infection. Structure-function studies on these molecules provide considerable insight into the mechanisms used by virus to evade the host immune system. Furthermore, these strategies might also be used to control dysregulated cytokine signaling found in autoimmune disease. See
1. Yoon et al. Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. J. Biol. Chem. 26586-26595, 2012 and
2. Nuara et al. Structure and mechanism of IFN-γ antagonism by an orthopoxvirus IFN-γ Binding Protein P.N.A.S. 105, 1861-1866, (2008).
Design of novel vaccines against human cytomegalovirus (HCMV): Current viral vaccines elicit antibodies to viral proteins that prevent/limit entry of virus into cells of their host. To date, this strategy has not been successful in a number of viruses, including HCMV, that directly target immune cell signaling pathways to evade host immune responses. HCMV infection can cause severe hearing loss, mental disabilities, and even death in a developing fetus. Currently there is no vaccine for HCMV. To address this problem, we are using our expertise in cytokine structural biology to design novel antigens that target virally produced cytokines. To date, these antigens show efficacy in animal models of HCMV. Further testing of this strategy and evaluating molecules for possible clinical trials are now underway. See
1. Logsdon et al. Design and Analysis of Rhesus Cytomegalovirus IL-10 Mutants as a Model for Novel Vaccines against Human Cytomegalovirus. PLoS One. 6 (2011).
2. Eberhardt et al. Host Immune Responses to a Viral Immune Modulating Protein: Immunogenicity of Viral Interleukin-10 in Rhesus Cytomegalovirus-Infected Rhesus Macaques. PLoSOne 7 (2012).