title: Small RNA analysis in Sindbis virus infected human HEK293 cells.
abstract: BACKGROUND
  In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells.
  
  
  RESULTS
  SINV AR339 and TR339-GFP were adapted to grow in HEK293 cells. Deep sequencing of small RNAs (sRNAs) early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral sRNAs (vsRNAs), with no size, sequence or location specific patterns characteristic of Dicer products nor did they possess any discernible pattern to ascribe to a specific RNAi biogenesis pathway. This was supported by multiple variants for each sequence, and lack of hot spots along the viral genome sequence. The abundance of the best defined vsRNAs was below the limit of Northern blot detection. The adaptation of the virus to HEK293 cells showed little sequence changes compared to the reference; however, a SNP in E1 gene with a preference from G to C was found. Deep sequencing results showed little variation of expression of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed no difference in expression by Northern blot analysis.
  
  
  CONCLUSIONS
  We show that, unlike SINV infection of invertebrates, generation of Dicer-dependent svRNAs and change in expression of cellular miRNAs were not detected as part of the Human response to SINV.
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich, Norfolk, United Kingdom.
authors.forename: Andras
authors.initials: A
authors.lastname: Donaszi-Ivanov
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom.
authors.forename: Irina
authors.initials: I
authors.lastname: Mohorianu
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom.
authors.forename: Tamas
authors.initials: T
authors.lastname: Dalmay
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich, Norfolk, United Kingdom.
authors.forename: Penny P
authors.initials: PP
authors.lastname: Powell
mesh_terms: D018354:Alphavirus Infections / Q000235:genetics* / Q000821:virology*; D015415:Biomarkers / Q000378:metabolism; D015152:Blotting, Northern; D020869:Gene Expression Profiling; D016679:Genome, Viral; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012845:Sindbis Virus / Q000472:pathogenicity*
chemical_list: D015415:Biomarkers; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral