Information on EC 2.1.1.375 - NNS virus cap methyltransferase

RNA-directed RNA polymerase L

VSV L

VSV L protein

2 S-adenosyl-L-methionine + G(5')pppAACA-[mRNA] = 2 S-adenosyl-L-homocysteine + m7G(5')pppAmACA-[mRNA]

overall reaction

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S-adenosyl-L-methionine + G(5')pppAACA-[mRNA] = S-adenosyl-L-homocysteine + G(5')pppAmACA-[mRNA]

(1a)

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S-adenosyl-L-methionine + G(5')pppAmACA-[mRNA] = S-adenosyl-L-homocysteine + m7G(5')pppAmACA-[mRNA]

(1b)

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S-adenosyl-L-methionine:G(5')pppAACA-[mRNA] N7,2'-O-methyltransferase

The enzyme from non-segmented negative strain (NNS) viruses (e.g. rhabdoviruses) catalyses two successive methylations. In higher eukaryotes the two methylations occur in the reverse order and are catalysed by two different enzymes (cf. EC 2.1.1.56, mRNA (guanine-N7)-methyltransferase, and EC 2.1.1.57, methyltransferase cap1) that do not required a specific motif.

2 S-adenosyl-L-methionine + G(5')pppAACA-[mRNA]

2 S-adenosyl-L-homocysteine + m7G(5')pppAmACA-[mRNA]

S-adenosyl-L-methionine + 13-nucleotide-long capped RNA

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S-adenosyl-L-methionine + G(5')pppAACA-[mRNA]

S-adenosyl-L-homocysteine + G(5')pppAmACA-[mRNA]

S-adenosyl-L-methionine + G(5')pppAmACA-[mRNA]

S-adenosyl-L-homocysteine + m7G(5')pppAmACA-[mRNA]

S-adenosyl-L-methionine + GpppGm(Am)-SUDV12

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Substrates: substrate for N-7-methyltransferase activity
Products: -

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S-adenosyl-L-methionine + mGpppA(Am)SUDV12

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Substrates: substrate for cap 2'-O-methyltransferase activity
Products: -

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S-adenosyl-L-methionine + mGpppGm-SUDV12

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Substrates: substrate for internal 2'-O-A methyltransferase activity
Products: -

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additional information

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2 S-adenosyl-L-methionine + G(5')pppAACA-[mRNA]

2 S-adenosyl-L-homocysteine + m7G(5')pppAmACA-[mRNA]

S-adenosyl-L-methionine + G(5')pppAACA-[mRNA]

S-adenosyl-L-homocysteine + G(5')pppAmACA-[mRNA]

S-adenosyl-L-methionine + G(5')pppAmACA-[mRNA]

S-adenosyl-L-homocysteine + m7G(5')pppAmACA-[mRNA]

S-adenosyl-L-methionine

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NaCl

maximum activity in presence of 150 mM NaCl

7

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30

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UniProt

Lyssavirus rabies SAD B19

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UniProt

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SwissProt

Sudan ebolavirus Human/Uganda/Gulu/2000

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SwissProt

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761770, 761771, 762265, 762505

UniProt

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761770, 761771, 762265, 762505

UniProt

Highest Expressing Human Cell Lines

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metabolism

Ebola virus - Eckron (Zaire, 1976)

physiological function

human respiratory syncytial virus

Escherichia coli K-12

14 entries

Henipavirus nipahense

human metapneumovirus

6 entries

Human respiratory syncytial virus A2

Marburg virus - Musoke, Kenya, 1980

Measles virus strain Ichinose-B95a

Respiratory syncytial virus A2

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structure of rabies virus L protein bound with its phosphoprotein cofactor, at 3.3 A resolution. In the methyltransferase domain, a shared GxGxG motif forms the binding site for the methyl donor, S-adenosylmethionine, and a conserved set of charged residues Lys1685, Asp1797, Lys1829, and Glu1867 forms the catalytic tetrad

Lyssavirus rabies

P16289

762290

domain VI of the VSV L protein contains a 2'-O-ribose methyltransferase (MTase) domain. Structural homology model of residues 1644-1842 within domain VI. The binding pocket consists of seven beta-strands and six alpha-helices. Highly conserved glycines are lining the interior of the pocket. Conserved aspartic acid D1762 is positioned at the perimeter of the pocket and interacts with invariantly conserved residues K1651, K1795, and E1833 to constitute the catalytic tetrad

the multifunctional large polymerase protein L is organized L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon viral phosphoprotein binding, L undergoes a significant rearrangement

F2113A

the mutation leads to more than 50% reduced N7-methylation, 2'O-methylation and internal methylation activities compared to the wild type enzyme

H2067A

the mutation leads to reduced N7-methylation, 2'O-methylation and internal methylation activities compared to the wild type enzyme

H2112A

the mutation leads to more than 60% reduced N7-methylation, 2'O-methylation and internal methylation activities compared to the wild type enzyme

K1813A

the mutation leads to a loss of the protein-RNA interaction compared to the wild type enzyme

K2043A

the mutation leads to about 30% increased N7-methylation activity and reduced 2'O-methylation and internal methylation activities compared to the wild type enzyme

K2118A

the mutation leads to severely reduced N7-methylation activity and no 2'O-methylation and internal methylation activities compared to the wild type enzyme

K2189A

the mutation leads to severely reduced N7-methylation activity and almost no 2'O-methylation and internal methylation activities compared to the wild type enzyme

K2189D

the mutation leads to severely reduced N7-methylation activity and no 2'O-methylation and internal methylation activities compared to the wild type enzyme

R2068A

the mutation leads to severely reduced N7-methylation activity and no 2'O-methylation and internal methylation activities compared to the wild type enzyme

R2120A

the mutation leads to wild type N7-methylation activity, and about 25% increased 2'O-methylation and about 25% decreased internal methylation activities compared to the wild type enzyme

R2165A

the mutation leads to increased N7-methylation, and reduced 2'O-methylation and internal methylation activities compared to the wild type enzyme

R2172A

the mutation leads to increased N7-methylation, and reduced 2'O-methylation and internal methylation activities compared to the wild type enzyme

R2193A

the mutation leads to severely reduced N7-methylation activity and no 2'O-methylation and internal methylation activities compared to the wild type enzyme

H2067A

Sudan ebolavirus Human/Uganda/Gulu/2000

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the mutation leads to reduced N7-methylation, 2'O-methylation and internal methylation activities compared to the wild type enzyme

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H2112A

Sudan ebolavirus Human/Uganda/Gulu/2000

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the mutation leads to more than 60% reduced N7-methylation, 2'O-methylation and internal methylation activities compared to the wild type enzyme

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K1813A

Sudan ebolavirus Human/Uganda/Gulu/2000

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the mutation leads to a loss of the protein-RNA interaction compared to the wild type enzyme

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K2043A

Sudan ebolavirus Human/Uganda/Gulu/2000

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the mutation leads to about 30% increased N7-methylation activity and reduced 2'O-methylation and internal methylation activities compared to the wild type enzyme

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R2068A

Sudan ebolavirus Human/Uganda/Gulu/2000

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the mutation leads to severely reduced N7-methylation activity and no 2'O-methylation and internal methylation activities compared to the wild type enzyme

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D1762A

D1762E

mutant is able to form plaques at 31°C in Vero cells with significantly reduced titers at 24 hr compared to wild-type

D1762G

mutant is able to form plaques at 31°C in Vero cells with significantly reduced titers at 24 hr compared to wild-type

D1762N

mutant is able to form plaques at 31°C in Vero cells with significantly reduced titers at 24 hr compared to wild-type

E1833Q

G1670A

mice inoculated with G1670A, which is defective only in G-N-7 methylation, are attenuated in vivo yet retain a low level of virulence

761771

G1672A

G1672P

mutant is able to form plaques at 31°C in Vero cells

G1674P

mutant is able to form plaques at 31°C in Vero cells, but not at 39°C

G1675P

mutant is able to form plaques at 31°C in Vero cells, but not at 39°C

G4A

mutant virus is completely defective in both G-N-7 and 2'-O methylation, exhibits low virulence in mice despite productive viral replication is not detected in lung and brain

761771

K1651A

K1795A

L1716T

mutant is able to form plaques at 31°C in Vero cells and displays a pronounced growth restriction at 39°C

L1716Y

mutant is able to form plaques at 31°C in Vero cells, but not at 39°C

D1762A

E1833Q

G1670A

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mice inoculated with G1670A, which is defective only in G-N-7 methylation, are attenuated in vivo yet retain a low level of virulence

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G1672A

G1672P

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mutant is able to form plaques at 31°C in Vero cells

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G1674P

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mutant is able to form plaques at 31°C in Vero cells, but not at 39°C

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G1675P

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mutant is able to form plaques at 31°C in Vero cells, but not at 39°C

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K1651A

K1795A

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mutation inhibits both 2'-O and G-N-7 methylations

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L1716T

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mutant is able to form plaques at 31°C in Vero cells and displays a pronounced growth restriction at 39°C

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54

melting temperature of the C-terminal domain

Co-NTA column chromatography and Superdex S75 gel filtration

expressed from recombinant baculoviruses in Spodoptera frugiperda 21 cells

expressed in Escherichia coli