Faculty: Dorothea Sawicki, PhD.

Summary: Dr. Sawicki's research effort is directed toward determining the molecular mechanisms governing RNA synthesis. The systems being studied utilize the alphaviruses Sindbis and Semliki Forest viruses. These as well as other Togaviruses are of interest because they produce disease in a variety of animals, including humans, and because they replicate in invertebrate as well as vertebrate animals. A cDNA clone of Sindbis that is capable of expressing infectious RNA genomes is being utilized to determine the role of the viral nonstructural proteins in the alphavirus replication cycle.

Encephalitic Viruses - Growth and Disease Mechanisms The laboratory studies basic replication features of alphaviruses, a group of enveloped RNA viruses transmitted by vector hosts (mosquitoes) to vertebrate hosts, such as birds, that serve as reservoirs of the virus in nature. They also infect horses and humans, and can cause significant brain (encephalitis) infections and inflammation (arthritis). Failure of the host innate and adaptive immune responses to prevent high levels of virus production allows invasion of the brain and the infection and death of neurons. A virus closely related to alphaviruses at the genetic level but transmitted between humans by inhalation of virus-containing respiratory droplets (coughing or sneezing by infected patients) is rubella virus, the agent of a measles-like exanthem (rash). Our work on alphaviruses will be of relevance to rubella as well.
Current research projects focus on: (1) identifying molecular mechanisms required to make RNA templates needed for virus production; (2) understanding what confers the stable nature of mature replication complexes used to make genomes and messenger RNA; and (3) defining how one of the viral proteins (nsP2) induces and controls antiviral responses by the host cell to allow replication before the cell dies. Loss of nsP2 functions results in a persistent infection, where infected cells survive as a source of infection to other cells and hosts. Discovering how viral proteins interact with host pathways and determine the outcome of virus infections could provide new strategies for antiviral drug design. Recent discovery of fish (salmonid) alphaviruses that target muscle tissue cells and are economically important and the recent epidemic emergence of Chikungunya virus disease in people in the Indian Ocean region underscore the medical importance of understanding alphavirus pathogenesis.

Figure 1		Figure 2
Eastern equine encephalitis virus, in mosquito salivary gland. This is an ultra-thin section of the salivary gland of an Aedes mosquito, showing large numbers of virions within the lumen of an upstream divereticulum of the salivary space. In this infection, large numbers of the 60 nm (nanometer) spherical virions crowd the salivary space, ready to be transmitted to the next vertebrate host when the mosquito seeks a blood meal. Magnification approximately x70,000. Micrograph from F. A. Murphy, University of Texas Medical Branch, Galveston, Texas.		Eastern equine encephalitis virus. This is an ultra-thin section of a Vero cell culture infected for 24 hours. Virions have accumulated in the space between cells as a result of budding from the surface membrane of infected cells. In this infection, very large numbers of the 60 nm (nanometer) spherical virions are produced quickly and as quickly the cells are destroyed. Magnification approximately x70,000. Micrograph from F. A. Murphy, University of Texas Medical Branch, Galveston, Texas.

My research interests focus on how viruses, particularly the mosquito-borne alphaviruses, infect and replicate in cells and how they alter the molecular environment of the host cells to accomplish their replication strategy. Specifically, this has involved the identification of viral and host factors essential for the successful replication of alphaviruses and their ability to cause disease. Alphaviruses are enveloped, arthropod-borne viruses whose genome is a single molecule of messenger-sense RNA and who are members of the Togaviridae, a virus family that includes the related, but non-arthropod-borne rubella virus, the cause of German measles. Alphaviruses are of general interest because they produce disease in a variety of animals, including humans, and because they replicate in invertebrates as well as in vertebrates. This broad reproductive potential is essential for their transmission and thus for the spread of their associated diseases. Alphaviruses cause persistent infection in mosquitoes, which are their reservoir in nature, and thus have evolved a mechanism that allows non-fatal viral replication in invertebrate cells. Infection starts with the translation and copying of the infecting genome into a complementary, or negative strand, RNA template and formation of a highly efficient and stable replication complex. My research interests focus how viruses, particularly the mosquito-borne alphaviruses, infect and replicate in cells and how they alter the molecular environment of the host cells to accomplish their replication strategy. Specifically, this has involved the identification of viral and host factors essential for the successful replication of alphaviruses and their ability to cause disease. Alphaviruses are enveloped, arthropod-borne viruses whose genome is a single molecule of messenger-sense RNA and who are members of the Togaviridae, a virus family that includes the related, but non-arthropod-borne rubella virus, the cause of German measles. Alphaviruses are of general interest because they produce disease in a variety of animals, including humans, and because they replicate in invertebrates as well as in vertebrates. This broad reproductive potential is essential for their transmission and thus for the spread of their associated diseases. Alphaviruses cause persistent infection in mosquitoes, which are their reservoir in nature, and thus have evolved a mechanism that allows non-fatal viral replication in invertebrate cells. Infection starts with the translation and copying of the infecting genome into a complementary, or negative strand, RNA template and formation of a highly efficient and stable replication complex. I investigate the mechanisms by which the alphaviruses Sindbis and Semliki Forest virus (SFV) regulate viral RNA synthesis to overproduce genome and messenger RNAs relative to their negative-strand template RNA. It is the synthesis of the negative-strand template that is the initial and essential step that starts viral replication with alphaviruses and other kinds of plus-stranded RNA viruses such poliovirus and hepatitis C virus to name two of many kinds that infect and cause disease in humans. I have demonstrated that alphaviruses are unique among positive strand RNA viruses because they regulate their synthesis of negative-strand templates, which occurs only within the first few hours of infection and then ceases. It is during this early period that viral replication-transcrption complexes (RTC) are formed. These RTC composed of minus strand template and viral proteins that are produced by translation of the genome and host proteins. After the RTC are formed they produce many copies of the genome and of subgenomic mRNA that are used to produce the structural proteins and that enclose the genome to produce infectious viral particlesThe long range goal of my research has been is to develop experimental methods that allow the investigation of the nature of the molecular mechanism by which alphaviruses create a RTC, how the host cells responds to the replication of the virus. Our study of alphavirus transcription has been funded by the NIH, National Institutes of Allergy and Infectious Diseases, beginning in 1978. Currently, we are investigating the structure and function of the viral nonstructural proteins in the replication of the genome and the transcription of the subgenomic mRNA and the temporal regulation of negative strand synthesis; the requirements for assembly of functional replication complexes for transcription and replication; and the role of host proteins and of polyprotein precursors of the nonstructural proteins in the formation of active alphavirus replication complexes. We have put forward the hypothesis that early in infection a new replication complex forms that contains a positive strand template and that is subsequently converted to one containing a negative strand template by the preferential binding of stable polymerases to the newly synthesized negative strand RNA, and that cessation of negative strand synthesis occurs because new replication complexes fail to be formed late in the replication cycle. Our recent results indicate the molecular mechanism(s) may include differential viral nonstructural polyprotein processing, specific polymerase modifications that alter promoter affinity for the two RNA templates and/or depletion of essential host factors.
The general research approaches may be summarized as follows. (1) Mutant gene segments are exchanged into an infectious Sindbis cDNA clone from which recombinant virus is prepared and assayed for retention of phenotype. (2) Mutations are located by performing sequencing, subcloning and construction of single mutation recombinant viruses to verify the functional significance of any base change. (3) Structural studies are carried out using site-directed mutagenesis. (4) Replication complexes are analyzed for activity in vivo and in vitro and for retention of viral and host components before and after detergent-solubilization. (6) Template-free replication complexes are purified and assayed for viral and host components and for template specificity. (7) An expression system is developed for reconstitution of transcriptionally active replication complexes.
In RNase L, but not PKR or Mx1 deficient mouse cells, Sindbis minus strand synthesis was continuous over a 10-12 h period, but the viral mature plus strand synthesizing replication complexes were no longer stable. Some of the RNase L cells became persistently infected, and this number varied with the confluency state of the monolayer. Such persistently infected cell cultures were sensitive to heterologous viruses (EMCV), confirming that interferon was not mediating the persistence. Our grant renewal now focuses on these findings and the nature of the host functions involved.
Dr. Sawicki was educated at Wells College, Auruora, NY, in biology, and at Columbia University, Department of Microbiology at the College of Physicians and Surgeons, in immunochemistry in the laboratory of Dr. Sam Beiser. Her postdoctoral training was in virology in the laboratory of Dr. Peter Gomatos at Memorial Sloan Kettering Institute. She joined the faculty in the Department of Microbiology at the University of Toledo College of Medicine (formerly the Medical College of Ohio) in 1977 and currently holds the rank of Professor. She has trained graduate and medical students, and was the director of the interdepartmental research training program in Molecular and Cellular Biology 1997-2007. She served as director of the reorganized graduate research training area of infection, immunity and transplantation from July 2007-July 2009. Current Grant Funding:

•	NIH-Regulation of alphavirus transcription and the role of the cellular RNase L endonuclease in alphavirus-host interactions