Adaptation of reovirus to growth in the presence of protease inhibitor E64 segregates with a mutation in the carboxy terminus of viral outer-capsid protein sigma3.
Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of sigma3 protein and proteolytic cleavage of micro1/micro1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 microM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein sigma3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of sigma3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of sigma3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in sigma3 protein containing amino acid 354 influences susceptibility of sigma3 to proteolysis during reovirus disassembly.
Departments of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
D H
DH
Ebert
J D
JD
Wetzel
D E
DE
Brumbaugh
S R
SR
Chance
L E
LE
Stobie
G S
GS
Baer
T S
TS
Dermody
D000222:Adaptation, Physiological; D000818:Animals; D002213:Capsid / Q000737:chemistry / Q000502:physiology*; D036022:Capsid Proteins; D002402:Cathepsin D / Q000494:pharmacology; D056668:Cathepsin L; D002403:Cathepsins / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D010450:Endopeptidases; D007739:L Cells; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D051379:Mice; D009154:Mutation; D016601:RNA-Binding Proteins; D012087:Reoviridae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D019065:Virus Assembly
D036022:Capsid Proteins; D015853:Cysteine Proteinase Inhibitors; D016601:RNA-Binding Proteins; C053179:sigma protein 3, Reovirus; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D002402:Cathepsin D; D007930:Leucine; C024974:E 64