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Characterization of temperature-sensitive mutants of human respiratory syncytial (RS) virus
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Caravokyri, Calliope (1990) Characterization of temperature-sensitive mutants of human respiratory syncytial (RS) virus. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3226316~S15
Abstract
Four ts mutants of RS virus, two from the subgroup A/wild-type strain A2 (ts A₁ ts A₂) and two from the subgroup B/wild-type strain RSN-2 (ts N₁, ts N₁₉), representing different complementation groups, were studied.
Mutant ts A₁ (complementation group A) was found to be restricted at a late stage of infection at 39°C. The degree of growth restriction was directly related to the length of incubation of ts A₁-infected cultures at 39°C, implying that the spread of infection (by cell-to-cell fusion) was affected. The SDS-PAGE profile of radiolabelled ts A₁ intracellular viral proteins at 39°C revealed a significant reduction in the amount of the F₁ polypeptide (the large subunit of the fusion protein), followed by a slightly less severe decrease of the matrix (M) protein. The cleaved but undissociated fusion (F₁,₂) protein of ts A₁ migrated more slowly than its wild-type counterpart on SDS-PAGE. Characterization of ts⁺ revertants revealed varying degrees of reversion to wild-type levels of growth at 39°C, but no coordinate restoration of the ts phenotype and of the ts A₁ F₁,₂ was observed. This suggested that the mutation affecting F₁,₂ mobility in ts A₁ could be compensated by secondary mutations in the same (F) or in another gene, or that the tsA₁ lesion is located in a different viral protein with which the F protein forms a complex during the virus cycle.
Mutant ts A₂ (complementation group B) was found to exhibit temperature-sensitive synthesis of the G glycoprotein which mediates RS virus attachment to host cells. Examination of the G-processing pathway showed that synthesis of the major p50 precursor was unaffected but further maturation to fully glycosylated G was defective at 39°C. Mutant ts A₂ also exhibited defective proteolytic cleavage of the Fₒ precursor into F₁ and F₂ subunits (a processing step required for creation of a fusion-active F protein) at both temperatures. This could explain the previously reported variability in plaque phenotype and growth of ts A₂ under permissive conditions in different cell types, since the proteolytic activation of the fusion protein is host-dependent.
Mutant ts N₁ (complementation group D) possessed an M protein which disappeared from the soluble cytoplasmic fraction of infected cells soon after its synthesis at 39°C. The ts N₁ M protein was also partially unstable at 33°C and exhibited slightly decreased SDS-PAGE mobility. The M protein of ts⁺ revertants was stable at 39°C, correlating the defect in M-stability with the ts N₁ phenotype. The M proteins of three ts⁺ revertants exhibited the slower mobility of the ts N₁ M protein, suggesting that they were pseudorevertants. The P protein of ts N₁ (and its ts⁺ revertants) also migrated more slowly than the wild-type RSN-2 P protein. Sequence analysis and in vitro expression of a ts N₁ P cDNA clone showed that the aberrant P mobility was due to a single amino acid change (Asn → Asp at position 217).
Mutant ts N₁₉ (complementation group E) was completely restricted at 39°C, indicative of a ts defect at an early stage of infection. The ts N₁₉ P protein lacked an epitope recognized by anti-P MAb 3-5. The P proteins of ts⁺ revertants, which exhibited wild-type growth at 39°C, had this epitope restored, confirming that loss of the P-epitope was correlated with the ts phenotype. Sequence determination of P cDNA clones, produced from RSN-2, ts N₁₉ and ts⁺ revertant P mRNA by reverse transcription and PCR amplification, identified a single amino acid difference (Gly → Ser at position 172) in the ts N₁₉ P protein. In vitro expression of the sequenced P cDNA clones and immunoprecipitation of the respective P proteins with MAb 3-5 showed that presence of the Gly₁₇₂ residue was required for MAb- binding. This residue was contained within a C-terminal domain of the P protein which could be independently expressed by internal initiation of in vitro translation at AUG₁₄₈.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Respiratory syncytial virus -- Genetic aspects, Microbial mutation, Paramyxoviruses, Viral proteins | ||||
Official Date: | June 1990 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Pringle, Craig R. | ||||
Sponsors: | Alexander S. Onassis Public Benefit Foundation | ||||
Format of File: | |||||
Extent: | xvi, 342 leaves : illustrations, charts | ||||
Language: | eng |
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