Human parainfluenza virus type 3 (HPF3), a member of the paramyxovirus family of non-segmented negative-strand RNA viruses, is an important agent of lower respiratory tract disease in children. This virus causes several of the most significant childhood viral diseases in both developed and underdeveloped areas of the world. Despite the medical importance of this virus, there are great gaps in our knowledge of the fundamental processes leading to growth of the virus, including the mechanisms of transcription and replication. Much of the biology of virus-host cell interactions, including the requirements for virus-induced membrane fusion and the propensity of this virus to cause persistent infections in both cell culture and animals (including man), remains obscure. The overall goal of this project is to elucidate the molecular mechanisms whereby HPF3 infects cells and causes cytopathology, produces progeny virus, and establishes persistence. This goal will be accomplished primarily by using well-defined in vitro systems to study the processes of membrane fusion, transcription and replication of the RNA genome, and persistent infection. In addition, a new in vitro system will be designed to study the role of cis-acting sequences in transcription and replication of the virus. The specific objectives of the current proposal are: (1) To determine the requirements for HPF3-induced membrane fusion, specifically by assessing the role of each of the two viral glycoproteins, the fusion protein (F) and the hemagglutinin-neuraminidase protein (HN). The induction of cell fusion at neutral pH, resulting in the formation of syncytia, is a characteristic feature of paramyxovirus infection in cell culture, and may be important in the pathogenesis of diseases caused by these viruses. The proposed studies will take advantage of the unique fusion properties of cells persistently infected with HPF3, and are a direct outgrowth of my demonstration that both the F and the HN proteins of HPF3 are required for membrane fusion. (2) To determine the viral and cellular proteins involved in transcription and replication of HPF3. For these studies, advantage will be taken of the well-defined in vitro transcription/replication assay system recently developed in my laboratory, a system which supports the complete transcription, replication and assembly into nucleocapsids of the HPF3 genome. (3) To analyze the specific viral nucleotide sequences that control viral RNA polymerase function. To achieve this, methods will be devised to assemble synthetic RNA into nucleocapsids in vitro. Specific sites on the viral sequence will be altered by restriction enzyme digestion and site-directed mutagenesis, to assess the roles of particular , viral genome sequences in nucleocapsid assembly and viral RNA polymerase function. (4) To extend the characterization of an in vitro model of HPF3 persistent infection, in order to analyze the factors responsible for persistence of this virus. The relative contributions of the viral genome and the host cell to the persistently infected phenotype will be assessed, and potential alterations in the stability and cell-surface expression of viral proteins in the persistently infected cells that may contribute to the maintenance of the persistent infection will be examined. These studies should lend insight into important molecular events in the life cycle of HPF3, and should assist in the design of future prevention and therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI031971-03
Application #
2066891
Study Section
Experimental Virology Study Section (EVR)
Project Start
1992-04-01
Project End
1997-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
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Porotto, Matteo; Yokoyama, Christine C; Orefice, Gianmarco et al. (2009) Kinetic dependence of paramyxovirus entry inhibition. J Virol 83:6947-51
Palermo, Laura M; Porotto, Matteo; Yokoyama, Christine C et al. (2009) Human parainfluenza virus infection of the airway epithelium: viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity. J Virol 83:6900-8