The assembly of the membrane of a lipid-containing bacteriophage, phi 6, will be studied as a means of elucidating the processes of membrane differentiation and translocation. Phi 6 contains a genome of three segments of double-stranded RNA inside of a ployhedral procapsid, which is in turn inside of another capsid shell. This double layered structure, called the nucleocapsid, is enveloped by a membrane that contains phospholipids and five species of protein that are specified by the phage genome. The assembly of the lipid-containing membrane on the particle is dependent upon the activity of protein P12, which is the only phage specified protein that is not a part of the virion. The assembly process has been studied in our laboratory in great detail using nonsense mutants of the phage. In addition, we have made cDNA clones of the entire genome, and we have sequenced the entire genome. We have also prepared expression vectors for both E. coli and the Pseudomonad hosts of the phage and demonstrated complementation activity of the cDNA material. We will investigate the assembly of the viral membrane by studying the interaction between protein P12 and nascent viral membrane proteins in vitro. Fusions of the membrane proteins with alkaline phosphatase will be prepared as a means of investigating whether particular parts of the proteins have targeting sequences. Physiological studies will also be performed to study the dependence of the localization of one or more of the membrane proteins on the presence of protein P12 and several other membrane proteins that may play important roles in the formation of the membrane. The formation of membranes and the transfer of membrane material is vital to many cellular processes such as organelle formation, receptor mediated responses, virus formation, and secretion of cellular materials. Understanding the processes would be of use in combating viral diseases, in ameliorating diseases due to dysfunction of hormone and metabolite receptor responses, nerve impulse transmission, cellular differentiation and proliferation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Public Health Research Institute
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Mindich, Leonard (2004) Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives. Virus Res 101:83-92
Onodera, S; Sun, Y; Mindich, L (2001) Reverse genetics and recombination in Phi8, a dsRNA bacteriophage. Virology 286:113-8
Qiao, X; Qiao, J; Onodera, S et al. (2000) Characterization of phi 13, a bacteriophage related to phi 6 and containing three dsRNA genomic segments. Virology 275:218-24
Hoogstraten, D; Qiao, X; Sun, Y et al. (2000) Characterization of phi8, a bacteriophage containing three double-stranded RNA genomic segments and distantly related to Phi6. Virology 272:218-24
Mindich, L (1999) Precise packaging of the three genomic segments of the double-stranded-RNA bacteriophage phi6. Microbiol Mol Biol Rev 63:149-60
Mindich, L; Qiao, X; Qiao, J et al. (1999) Isolation of additional bacteriophages with genomes of segmented double-stranded RNA. J Bacteriol 181:4505-8
Mindich, L (1999) Reverse genetics of dsRNA bacteriophage phi 6. Adv Virus Res 53:341-53
Qiao, X; Qiao, J; Mindich, L (1997) An in vitro system for the investigation of heterologous RNA recombination. Virology 227:103-10
Onodera, S; Qiao, X; Qiao, J et al. (1995) Acquisition of a fourth genomic segment in bacteriophage phi 6, a bacteriophage with a genome of three segments of dsRNA. Virology 212:204-12
Qiao, X; Qiao, J; Mindich, L (1995) Interference with bacteriophage phi 6 genomic RNA packaging by hairpin structures. J Virol 69:5502-5

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