Tamer Salem, Verena Lietze, Alejandra Garcia-Maruniak, James Maruniak, and Drion Boucias

Transcript Analysis of the House FlyVirus, Musca domestica Salivary Gland

Hypertrophy Virus (MdSGHV)

- enveloped- dsDNA- circular- 124,279 bp

DAPI/Phalloidin Staining

Control SGHV-5 Days

Virus Structure

R T C h x b n G W Q s r S q X J

dr1 dr2 dr3,4

Z f j

w z

L u H M U I P t E

y dr8 dr9

A B a v e i k K

dr11 dr12 dr15

O p l d O g D m F V N Y c R

1 dnapol

2 3 4

5 9

6 7 8 10 11mcp dhfr

12 ts

13

14 15 16 17

18

2119 20 2822 23 24

27

29pif-1

25 26

30 31

Baculovirus and Nudivirus homologues

32 33atpase

35 36 MMP

37 38 39p74

41

34 40 42

43

44

45

4647

odv-e66

48 49Ac150

50Aminoacylase-1

51

52dutpase

53 54 55 56 57 58

59 60 6162rr2

63 65rr1

64nucleoside cotransporter

66

67

68

69tk

70 71

72 73 74 75 76

Nudivirus homologues

105

77 78iap

79 80

81

82integrase

83

84

85 86

87 88 89pif-2

90

91 92 93 94

95

9697 98

99

100 101

102 103

104Helicase

106pif-3

108Ac81

Herpesvirus homologues

Direct repeats (dr) & TCA repeat

Cellular Homologues

Baculovirus homologues

dr5 dr6,7

dr10 dr13, 14

dr16 dr17 TCA dr18

107

Garcia-Maruniak et al., 2008

108 putative ORFs

- sensitive.- 3'-UTR.- sequencing RACE products.

CDS 3‘ UTR AAA…..AAAn5’ UTR

Forward primer

Rapid Amplification of cDNA 3' Ends(3'- RACE)

ORFs match in silico w/ 3’UTR (94 ORFs).

ORFs validated as a transcript by RT-PCR (13 ORFs).

ORFs not detected (ORF 41).

B a v e i k K

dr15

1 dnapol

12 ts 14 15 16 17 2822 23 24

29pif-1

35 36

MMP

53 54 55 56 57 58

72 73 74 75 76 77 78iap

79 80

81

82integrase

83

84

104Helicase

106

pif-3

R T C h x b n G W Q s r S q X J

dr1 dr2 dr3,4

2 3 4

5 9

6 7 8 10 11mcp dhfr

13 18

2119 20

27

25 26

30 31

Z f j

w z

L u H M U I P t E

y dr8 dr9

32 33atpase

37 38 39p74

41

34 40 42

43

44

45

4647

odv-e66

48 49Ac150

50Aminoacylase-1

51

52dutpase 59 60 61

62rr2

dr5 dr6,7

O p l d O g D m F V N Y c R

105

85 86

87 88 89pif-2

90

91 92 93 94

95

9697 98

99 102 103 108Ac81

dr16 dr17 TCA dr18

107

65rr1

69tk

A

dr11 dr12

63

64

67

68 70 71

dr10 dr13, 14

100 101

ORFs Validated

ORFs share the same transcript (14 ORFs).

ORFs show splicing (ORF 45).

B a v e i k K

dr15

1 dnapol

12 ts 14 15 16 17 2822 23 24

29pif-1

35 36

MMP

53 54 55 56 57 58

72 73 74 75 76 77 78iap

79 80

81

82integrase

83

84

104Helicase

106

pif-3

R T C h x b n G W Q s r S q X J

dr1 dr2 dr3,4

2 3 4

5 9

6 7 8 10 11mcp dhfr

13 18

2119 20

27

25 26

30 31

Z f j

w z

L u H M U I P t E

y dr8 dr9

32 33atpase

37 38 39p74

41

34 40 42

43

44

45

4647

odv-e66

48 49Ac150

50Aminoacylase-1

51

52dutpase 59 60 61

62rr2

dr5 dr6,7

O p l d O g D m F V N Y c R

105

85 86

87 88 89pif-2

90

91 92 93 94

95

9697 98

99 102 103 108Ac81

dr16 dr17 TCA dr18

107

65rr1

69tk

A

dr11 dr12

63

64

67

68 70 71

dr10 dr13, 14

100 101

ORFs found on the same transcript

GTATCTTGGGTTTGGGCTGAACCACCATCACCCATATAGGTAAGACATGAACTTGTCCGATTATC26 bases ORF 3 and 4

ORFs out of frame

ORFs in-frame

TTCCATTCGCAACAAGAATTAAGTGAACTCGCATTCTAACAAAATGTCCCACTACGACGCAGCTA21 bp

ORF 61 and 62

ORFs have both

CAGTCGTCTCCTCAAAGTAGGGACACACATACATATCACCCCCCATCATGAGTCTTGCATTGCAT27 bpORF 24, 25 and 26

CCGCCGTATTCATCTATAGATAATTACTATGGCTAAAGCGAGTCTTGATTCAATCTCACGCAATA2 bp

ATGAAGATTAATATAAGATTACTATATAGTCCAACTTCCGATCGCTGCCACCACCAC ORF 84

AGAACTAATAAAATAAGAAAGAAACAAATGAGAAAAATTTTTGTTTTGACTCAATTA ORF 84/85

28sORF 84

ORF 84/85

ORF 85

C 1 2 dpi

C 1 2 dpiB RT-PCR

ORF 85ORF 84 AAA…..AAAn

ORF 84 AAA…..AAAn

84F 84R 85F 85R

Temporal Transcription

ORF 84/85

ORF 84

3'- RACEA RT-PCR

84F

84R

28sF28sR

84F

85F

85R

U2 (major Class) 5’ splice site

MAG GT RAGT (M= A or C,R= A or G)ATG GT GAGT

IntronExon

U2 (major Class) 3’ splice site

C AG G T AG A

ExonIntron

Splicing

ORF_45 3‘ UTR

ORF_43

ORF_44

1143 bases 13 bases 262 bases847 bases

936 bases

ATG

ATG

1110 bases

138 bases

21 bases

CDS 3‘ UTR5’ 3’

ORF #Unidirection⇒⇒ or ⇐⇐

Convergent⇒⇐

Divergent⇐⇒

1 2 478 bases

2 3/4 264 bases

1 3/4 13 bases

17 18 111 bases

21 22 32 bases

22 23 120 bases

23 24/25/26 80 bases

24/25/26 27 9 bases

28 29 80 bases

29 30 64 bases

30 31/32 4 bases

34 35 2 bases

37 38 45 bases

42 43 102 bases

43 44 52 bases

ORF #Uni.

⇒⇒ or ⇐⇐Con.⇒⇐

Div.⇐⇒

48 49 9 bases

49 50 147 bases

50 51 101 bases

52 53/54 35 bases

61/62 63 23 bases

66 67 105 bases

77 78 1 bases

79 81 *25 bases

82 83 *5 bases

86 87 2 bases

89 90 32 bases

90 91/92 59 bases

94 95 85 bases

98 99 68 bases

100/101 102/103 128 bases

107 108 *25 bases

Uni. Con. Div.11 17 3< > Uni. Con. Div.>>

Mammalian

Sense-Antisense Gene Pairs (S-AS)

GGCAAGCTGAGATTTATTTACATAATAAATCAGTGCATTGTCTAAAAAAATCACTGCCTTTTG

GTTCTTAATGGTGTACGGCATGATTAATAAAGATTTTGCAATTTAATAAAAGTTTTTGTGGGGG

AATAAA DSEPoly(A) site

U/GU-richPy(A)n

17-41

Heterogeneity in Polyadenylation

GCTTAGCTACTAAATAAAATACACTCTCTAACATATTCTATGGTGTTTTTATTGACAATTAATTA

Stop codonAATAAA

DSEPoly(A) site

U/GU-richPy(A)nStop codon

Stop codon

ATTGTGAGATTTATGAAAAATACAATAAATGTTTCACTTATGGGCTAACATGTTGGTGTTGATG

TGTCTACATACTAATAAATTTTCTATGCTACTGGATATTCAATCGGGATAAGAGTCCTTTTATTTT

TCGATATTCCAAACATACCAATAATAAATAAAGCATTTCTGATTCCTAAAAATATTGTCCAATTATTAATTACAATGATTGACTTCTTGGGCAAAATAAAAATTAAAATTACAATAAAAATTACAGCGTCTATAACCCGAAATAAAAAACCAAATATATATCTTCTTTTATCGCTTTATTCTAAAAATATACATGTTT

AATAAA DSEPoly(A) sitePoly(A) site

AATAAA DSEPoly(A) siteAATAAAATTAAAAATATA

DSEPoly(A) siteATTAAAAATATAAATACA

GATGATGGTCTGGCTATTAAATACAACAAACTAATGTTTAATTTCGACAAAATTTTGGCATTCAATATGATGTAGTAAATGAATGCAAACAATATATTCGTTCGTCTGGAATTGTGGTCTTTAAAGACGCGAGTCTTGAATACCTCCCTATAGTCAAATACACTTTCATTCAAAATTGGTAATTTGT

Summary

• 107 out of 108 putative ORFs have been validated

• ORF 45 showed splicing in the 3’UTR

• 20 sense-antisense pairs (1 over 400 bases,6 over100 bases)

• polyadenylayion showed heterogeneity which mayplay a role in viral temporal gene expression

• Proteomic analysis

• Temporal transcription

• RNAi

Ongoing Research

Acknowledgement

Technical supportWilliam Kipperstock UFl Department of EntomologyNii Sai Torto UFl Department of EntomologySavita Shanker (ICBR Sequencing Core)Chris Geden, Melissa Doyle (USDA, CMAVE)

Financial SupportNIH/NIAID

ORF90

ORF 52

ORF 66

T T TA T T C T A A A A A TA T A CATGTTTTTGTTTATTATCTT T T A T T C T AT T TA T T C T A A A A A TA T A