Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Viruses
2.2. Illumina Deep Sequencing
2.3. Molecular Models
3. Results
3.1. Deep Sequence Analyses of Reference HHV-6A Strain U1102 Shows Corrections
3.2. Changes with BACmid Cloning and Reconstruction
3.3. Emerging Variant Genome Populations Generated after Tissue Culture Passage
3.4. Virus Envelope Genes Mutations from Tissue Culture Passage Affect Fusion Complex
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Amplicon | Forward Primer (5′-3′) | Reverse Primer (5′-3′) | Size (bp) |
---|---|---|---|
DR1 | ACAACCGCATCTTCTTCAG | ATGAACGGAGATCTGGGAG | 1750 |
DR1DR6 | CCTCATCTGTTATTCCCTTCC | ACTCACCGCAGTCTTGAAC | 2872 |
DR6 | TCCAAACTGTACGGCTATG | TGGCGTCTAATCCAGGTAC | 1858 |
DR_UL | CATGAGAGGACACTGGACGTACTC | GACATCTGTGGACCATGCA | 2339 |
0203 | ACCATGTGCGGAGAAAGTTG | ACGAGAATGAATATGCCGATG | 2174 |
0306 | ACTCCTATCCGTCTAAACTGCT | ATTTCTCACGCCGGTATTC | 4417 |
0611 | ATGCCAAATCTAGCTGCTG | GAATCGGAGGAAGCTAACGT | 4757 |
1114 | TCTTCAGGTGTCTCAACCTTC | TGATGATCTGCTGTTGGAAC | 5737 |
1419 | TTGAGATGCAGAGCCTCTC | ACGTGGTGGATAATTCTTCAG | 5370 |
1923 | CTACATCATCGGTTGCCATA | TACAGAAGTTTCGGCAACTG | 5216 |
2328 | TAGTCGCTGGATGTCGATAC | GAGGAATTGTGGACAACGCT | 4721 |
2830 | CATCCAACTCCTTGTCCGT | TCGTTCGACGTGTCTGAC | 5753 |
3031 | GACACTGTCCAACGCATCT | CTCTTGGGCATAAGTCCAGTA | 6552 |
3134 | ACAGAGCGACACCCAATATTC | AGCTGTGTCGATGTCTTGCTA | 2787 |
3439 | TGCATTCTCTCACAGCATG | TGTTTCCTTATGCCACCAATC | 6309 |
3941 | TGAGGCATGAGACTCAGAAG | AGCGGCATCCATAATGCTTA | 4796 |
4142 | AGCATCGTTTGAAGACACC | GAGTTGTTGTTCGCTGGTCT | 2810 |
4142INT | TTGTTTCAGGCCAGCATC | ATACCGAAAACTCAGGCAAC | 2983 |
4245 | AGACCGCATTCGTATCGCT | CACGATCAAGTTCGAATCGTA | 5092 |
4549 | GAGTGCTCGGATAAGTTCATT | ACGATACAATCCGGAATGC | 5922 |
4953 | TGGAACTCGGAATTGTTTCT | GTTTGAGACTGTATCTGCGCA | 4282 |
5357 | TATGGTGAAGAACCGTCG | TCCGCAGATAACAGAATGG | 4707 |
5758 | CAGTGCCATCTGTTATGAATAG | AGTCGCAGTAAGGTCCACGT | 6792 |
5864 | TGCTGAAATTACCTCAGTGAG | CCATTCAGTATAATGCATCC | 4229 |
6469 | CTGTATCAATATCAAGGCGG | ACCACTGAATACGAACGCTC | 5111 |
6974 | GCGATTGTTCTGCGATAGAG | CTTATATCCGAGCCTTGCAGT | 6472 |
7476 | GAGAACATGCTAGACAATTGG | CAAGAACTGCGACTCAATCC | 2668 |
7679 | GTTGCTAGTTGTATGACTTGG | CAATATCCACCGTTAGAGAAC | 7681 |
7984 | AGATGTATGCTGAAGAACGTG | GGATCGTCAACCGTTAGTG | 5109 |
8486 | CACAGTGTGTTCGCCGGAAG | ACAGACAATGCACATCCTCTG | 4773 |
8690 | AGGTTGATACGGCAACGAG | GCAATCATTAGCATACAGATG | 3326 |
8690INT | TGAGGAATCACGTGTTTG | ATCGAATCTATCCATGAAGATG | 1746 |
90R3 | CATCATTGTTATCGCTTTCAC | GCAACCGCAGTTCCTGTT | 4912 |
R395 | GCGGTACCCACTGATCTT | AGTCTACCAGGCATTCCGT | 6250 |
95100 | GAGGAGGGTCTGTCTAGATGT | TCGGAGATATCATAATCTGCGT | 4031 |
100DR | TTATAGTTGCTCCCGAAAGC | AAGAAGATGCGGTTGTCTTG | 2358 |
ORF or Locus | Refseq bp | Refseq:SNP | Non-Synonymous SNP Amino Acid Change, Codons and (Reverse Complement Codons) | This Study or as Cited |
---|---|---|---|---|
DR-L DR6 | 6663 | G:GG | ||
“ | 6665 | G:GG | Frameshift extension | [16] |
DR-L repeat | 7623 | G:C | “ | “ |
Repeats left end U | 8262 | AGGAG:A | ||
“ | 8362 | C:CGCG | ||
“ | 8518 | T:C | ||
“ | 8559 | ACAACA:CAC | ||
“ | 8591 | AT:A | ||
“ | 8602 | GG:G | ||
U10 | 18884 | C:Indel1 | Frameshift extension | [34,35] |
U40 | 63871 | G:T | Thr:His (ACG:CAG) | |
“ | 63872 | T:G | “ | |
U42 | 70446 | A:ATC | HisCys:GlnLeuAsp (CACTGT:CAACTGCATG) | Jones & Teo 1993 in Genbank L20954 |
“ | 70449 | G:GT | “ | Jones, M. in Genbank X92436 |
U57 mcp | 92149 | C:G | Lys:Asn ( AAG:AAC) | |
U58 tbp | 94068 | A:G | Met:Val ATG:GTG | |
U83 chemokine | 123740 | C:G | AspGlu:GluGln GACGAG:GAGCAG | [15] |
“ | 123741 | G:C | “ | “ |
U86 IE2 | (128132) | (Indel G) | Frameshift U86/U87 | In RefSeq; [17] |
R1 repeats | 128325 | C:T | ||
“ | 128443 | C:T | ||
“ | 128914 | G:A | ||
“ | 129917 | A:ATCC | ||
R2 repeats | 132312 | GG:G | ||
R3 repeats | 137831 | C:G | ||
“ | 137832 | G:C | ||
“ | 137906 | C:G | ||
“ | 137907 | G:C | ||
U100 | 148154 | C:G | ||
“ | 148155 | G:C | ||
U100 gQ1 | 149553 | C:G | Arg:Ala (CGT: GCT) | |
“ | 149554 | G:C | “ | |
DR-R DR6 | 157898 | G:GG | Frameshift extension | [16] |
“ | 157900 | G:GG | “ | |
DR-R repeat | 158857 | G:C |
ORF or Loci | RefSeq bp | RefSeq :SNP | Amino Acid Change, Codons and (Reverse Complement Codons) | % New Reference | % BAC Virus | % Passaged Virus | Comment, Citations, and Other Strains with SNPs |
---|---|---|---|---|---|---|---|
DR | 8016 | A:C | - | 87% | - | End T2 | |
“ | 8077 | C:CGA | 96% | - | - | HHV-6A AJ KP257,584.1, GS KC465,951.1; CiHHV6A 2284 KT895,199.1, 1501 KT355,575.1; [37]; Sites cleavages after pac2; [26,31] | |
“ | 8080 | G:GAC | - | 86% | - | CiHHV6A 2284 KT895,199.1, 5055, 5814 [16,24]; “ | |
Uleft repeat | 8157 | G:GC | - | - | 78% | ||
“ | 8558 | A:AC | 88% | AAC:A 99% | 90% | AC repeat | |
“ | 8561 | AAC:A | - | 100% | 98% | HHV-6A AJ [15,31] | |
“ | 8601 | CG:C | 97% | 87% | 86% | ||
U2 | 8876 | G:T | Ala314:Glu(GCG:GAG) | - | - | 53% | |
U19 UTR | 28,371 | C:CA | - | 52% | 72% | HHV-6A AJ | |
U31 tegument | 45,667 | A:T | Asn173:Ile AAC:ATC | - | 73% | 96% | |
U33 capsid | 52,148 | A:G | Tyr330:His(TAT:CAT) | - | 54% | 69% | |
U39 gB* | 61,162 | T:C | Thr193:Ala(ACG:GCG) | - | 58% | 99% | |
U41 dbp | 64,707 | C:T | Ala972:Thr (GCT:ACT) | - | - | 90% | HHV-6A GS KJ123,690.1 |
ORI repeat | 67,674 | T:C | - | - | 54% | ||
“ | 67,860 | T:C | - | - | 34% | HHV-6A GS KJ123,690.1 & KC465,951.1 | |
U50 capsid | 81,583 | G:A | Ala258:Thr(GCA:ACA) | - | 59% | 78% | HHV-6A GS KC465,951.1 |
“ | 82,158 | G:C | - | 67% | - | ||
U73 obp | 109,214 | G:A | Ser297:Asn(AGT:AAT) | - | - | 99% | |
U83 chemokine | 123,504 | G:GTT | f/s N-terminal extension | <1% | - | - | cDNA clone [13,14,15]; HHV-6A AJ KP257584; CiHHV6A: 5055 AIX09949, 23,090 AIX09956, 1501 KT355,575 |
U86 IE2 | 126,126 | T:C | Asn395:Asp(AAT:GAT) | - | - | 99% | |
“ | 127,092 | C:CTGA | Ser976:SerSer (TCA repeat ×9:×10) | - | 45% | - | 2 Ser insertion CiHHV6A 5055; 5814; [24] |
R2 repeat | 131,092 | C:T | - | - | 56% | IE2 intron | |
“ | 131,743 | G:C | - | - | 52% | “ | |
“ | 131,745 | A:C | - | - | 62% | “ | |
R2 region | 132,303 | TG:T | - | 64% | 56% | “ TGG:T HHV-6A AJ [31] | |
R3 repeat | 140,872 | G:A | - | 96% | - | End 30× repeats from 137,775; latency IE1/2 intron [38] | |
U95 | 142,969 | T:C | Trp10:Arg TGG:CGG | - | - | 54% | |
“ | 142,979 | T:A | Ile13:Asn ATT:AAT | - | - | 53% | |
“ | 143,591 | C:A | Ser217:Tyr TCC:TAC | - | - | 76% | |
U100 gQ2 | 146,729 | C:T | C-terminal truncation; Trp186:stop (TGG:TGA) | <1% | - | - | [12]1 (in 7/8 gQ2 cDNA clones HHV-6A U1102) |
“ | 146,862 | G:A | Ser42:Leu (TCG:TTG) | - | 60% | 99% | Not in HHV-6A HHV-6A U1102 from [39] |
U100 gQ1 | 149,251 | Indel2:A | intron | - | 44% | 83% | HHV-6A AJ; CiHHV6A 2284, 5055, 5814 [24] |
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Tweedy, J.G.; Escriva, E.; Topf, M.; Gompels, U.A. Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes. Viruses 2018, 10, 16. https://doi.org/10.3390/v10010016
Tweedy JG, Escriva E, Topf M, Gompels UA. Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes. Viruses. 2018; 10(1):16. https://doi.org/10.3390/v10010016
Chicago/Turabian StyleTweedy, Joshua G., Eric Escriva, Maya Topf, and Ursula A. Gompels. 2018. "Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes" Viruses 10, no. 1: 16. https://doi.org/10.3390/v10010016