Summary auto-generated
This study examines how mutations in the 5' noncoding region (5'NCR) of hepatitis A virus (HAV) strain GBM influence viral growth in different cell types. Researchers observed that when GBM virus was passaged in human embryonic lung fibroblast (HFS) cells, specific mutations accumulated in the 5'NCR (at positions 153, 178, 646, and 687), whereas no 5'NCR mutations occurred during passage in fetal rhesus monkey kidney (FRhK-4) cells. To test the importance of these mutations, the authors constructed chimeric viruses containing different 5'NCR sequences from GBM variants within an HM175 viral backbone. Transfection experiments revealed that the 5'NCR sequence is critical for HFS cell growth but less important for FRhK-4 cell growth. Only the chimeric virus containing the HFS-adapted 5'NCR sequence (with all four mutations) could produce infectious virus in HFS cells. In contrast, multiple different 5'NCR variants supported viral growth in FRhK-4 cells. The results demonstrate that the 5'NCR plays a key role in cell-line-specific adaptation of HAV, particularly determining host range and replication efficiency in different primate cell types.
Key findings
- The 5'NCR of HAV strain GBM undergoes specific mutations (A153G, A178G, G646T, T687G) when adapted to growth in HFS cells, but no 5'NCR mutations occur during passage in FRhK-4 cells
- The 5'NCR sequence is essential for HAV growth in HFS cells; only chimeric viruses containing the HFS-adapted 5'NCR can replicate in these cells
- The 5'NCR has minimal influence on HAV growth in FRhK-4 cells; multiple 5'NCR variants support comparable viral replication in this cell line
- The 5'NCR acts as a determinant of host cell range and viral adaptation, controlling cell-type-specific replication efficiency independent of other genomic regions
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Abstract
Previous sequence analysis of consecutive passages of the hepatitis A virus (HAV) strain GBM/WT in human embryonic kidney cells (HEK cells), human embryonic lung fibroblasts (HFS cells) and in FRhK-4 cells (foetal rhesus monkey kidney cells) pointed to a host cell dependent cell culture adaptation of GBM/WT in HFS cells involving mutations in the 5' noncoding region (5'NCR). Multiple nucleotide changes occurred in the 5'NCR of the GBM genome after the cell line used for virus passage was changed from HEK cells to HFS cells. In contrast, no mutations in the 5'NCR occurred during the first 20 passages of GBM/WT in FRhK-4 cells. In order to analyse the influence of the 5'NCR on host cell specific adaptation of HAV strain GBM in different cell cultures, GBM/HM175 chimeras were constructed which contained 5'NCRs from different GBM variants by replacing the 5'NCR of the infectious clone pHAV/7. Parallel transfection assays in FRhK-4 and HFS cells, performed with transcripts from the chimeric GBM/HM175 constructs, showed that the 5'NCR of the GBM variant GBM/HFS is essential for virus growth in HFS cells. The GBM/HM175 chimeric RNA, which contained the 5'NCR of GBM/HFS, exclusively, was able to produce infectious virus after transfection of HFS cells. The growth of the different GBM/HM175 chimeras in FRhK-4 cells, in contrast, did not seem to be strongly influenced by a specific sequence of the 5'NCR.