Inhibition of viral RNA methylation in herpes simplex virus type 1-infected cells by 5' S-isobutyl-adenosine

Cycloleucine, a competitive inhibitor of ATP: L-methionine S-adenosyltransferase in vitro, has been used to reduce intracellular concentrations of S-adenosylmethionine and by this means to inhibit virion RNA methylation in chicken embryo cells that are infected with B77 avian sarcoma virus. Under conditions of cycloleucine treatment, where virus production as measured by incorporation of radioactive precursors or by number of infectious particles is not significantly affected, the internal m6A methylations of the avian sarcoma virus genome RNA are inhibited greater than 90%. The predominant 5'-terminal structure in viral RNA produced by treated cells in m7G(5')pppG (cap zero) rather than m7G-(5')pppGm (cap 1). It appears from these results that internal m6A and penultimate ribose methylations are not required for avian sarcoma RNA synthesis and function. Furthermore, these methylations are apparently not required for transport of genome RNA to virus assembly sites. The insensitivity of the 5'-terminal m7G methylation to inhibition by cycloleucine suggests that the affinity of S-adenosylmethionine for 7-methylguanosine methyltransferase is significantly greater than for the 2'-0-methyltransferases or the N6-methyltransferases.

Section: Discussionmentioning

confidence: 99%

Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA

Dimock¹,

Stolzfus²

1978

“…While this manuscript was in preparation, Jacquemont and Huppert (1977) published evidence for inhibition of viral RNA methylation by another SAH analogue, S-isobutyladenosine. In contrast to STH, S-isobutyladenosine is a poor inhibitor of methylases in vitro (Legraverend et al, 1977) and thus the basis for its action in vivo is not clear.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies on the function of mRNA methylation have focused on the role of the cap structure in vitro at the translational level, using cell-free protein-synthesizing systems (Muthukrishnan et al. 1975;Both et al, 1975a), ribosome binding studies (Dasgupta et al, 1975;Both et al, 1975b;Kozak and Shatkin. 1976).…”

mentioning

confidence: 99%

In vivo inhibition of Novikoff cytoplasmic messenger RNA methylation by S-tubercidinylhomocysteine

Kaehler¹,

Coward²,

Rottman³

1977

The analogue S-tubercidinylhomocysteine (STH) has been used to study the methylation of mRNA in vivo. Partial inhibition of cytoplasmic poly(A)-RNA methylation was observed using a level of inhibitor which still permitted cell growth. Characterization of the partially methylated mRNA indicated the presence of cap structures lacking 2'-O-methylnucleosides, m7GpppN', which are normally not found in mammalian mRNA. Inhibition of additional methylated sites in mRNA at the second 2'-O-methynucleoside, and at internal N6-methyladenosine was also observed Methylation of 7-methylguanosine was not affected under the conditions used in these experiments. The methylnucleoside composition of cap structures differed in STH-inhibited and uninhibited cells. These results indicate that a completely methylated cap is not required for transport of mRNA into the cytoplasm. Furthermore, it may now be possible to assess in vivo the sequential nature of mRNA methylation and its potential role in mRNA processing.

“…Methylation of mRNA in vivo has been shown to be inhibited by cycloleucine (Bachellerie et al, 1978;Dimock & Stoltzfus, 1979), 5-tubericidinylhomocysteine (Pugh et al, 1977;Kaehler et al, 1979), and S-isobutyladenosine (Jacquemont & Huppert, 1977). However, methyl-deficient mammalian mRNA, a substrate for mRNA methyltransferases, has not been isolated heretofore.…”

Section: Discussionmentioning

confidence: 99%

Accumulation of methyl-deficient rat liver messenger ribonucleic acid on ethionine administration

Goswami¹,

Sharma²

1980

Highly purified poly(adenylic acid)-containing RNA isolated from livers of rats fed 0.25% DL-ethionine in the diet for 7 days accepted methyl groups from S-adenosyl[methyl-3H]methionine, when incubated in vitro with mRNA methyltransferases from vaccinia virus or Ehrlich ascites cells, whereas RNA from control rats had no such activity. Nuclease digestion followed by chromatographic analyses of mRNA methylated in vitro revealed that the methyl groups were incorporated at the 5' end into cap 1 structures (m7GpppNmp...) by the viral enzyme, whereas both cap 0 (m7GpppNp...) and cap 1 (m7Gpppm6Am...) structures were formed by the Ehrlich ascites cell enzymes. The methyl-deficient mRNA isolated from the liver of ethionine-fed rats differed in its translational properties from mRNA isolated from control animals in an in vitro protein synthesizing system from wheat germ.