Transcriptional map of the measles virus genome

50

SYNTHESIS

OF INFLUENZA

VIRAL GENOME

TEMPLATES

IN VITRO -__

ANN R. BEATON and ROBERT M. KRUG Sloan-Kettering Institute, 1275 York Avenue, New York, N.Y. 10021 U.S.A. To elucidate the mechanism(s) of influenza viral RNA replication, we have developed an -_I in vitro system that catalyzes the synthesis of genome template RNAs i.e., full-length transcripts of the virion RNAs (vRNAs). This synthesis involves two changes from viral mRNA synthesis: 1) initiation without a primer and 2) antitermination at the poly(A) addition site. We have succeeded in preparing extracts active in template RNA synthesis from nuclei of infected cells. Synthesis of both viral mRNAs and template RNAs by these extracts was stimulated lo-50 fold by the addition of the dinucleotide ApG, which has been shown to initiate transcription exactly at the 3' ends of the vRNA templates. This circumvention of the normal initiation step that occurs during template RNA synthesis facilitates the determination of the mechanism Fractionation of the nuclear extracts by high speed cenof antitermination. trifugation yields a pellet fraction which is active in the synthesis of viral mRNAs, but not of template RNAs, and a supernatant fraction which has minimal activity in either viral mRNA or template RNA synthesis compared to that of the pellet. When the two fractions are mixed together, template RNA synthesis is restored to the pellet, indicating that the supernatant contains a factor that The antitermination step is also dependent on is required for antitermination. the nature of the initiation step. When a capped RNA rather than ApG is added as primer, only viral mRNA and not template RNA synthesis occurs. This ensures that only the full-length genome templates, and not the mRNA transcripts containing capped fragments derived from host cell RNAs, are usedas templates for replication.

51

TRANSCRIPTIONAL

MAP

OF

THE

MEASLES

VIRUS

GENOME

PETERDCsWLING,EX%I7@lINBLUMHERG, JOSEPHMENONNA, JEANADAMUS, DANIEL KOLAKOFSKY and STUART co3K. Neurosci. Dept., UMDNJ-New Jersey Medical School, Newark 07103, U.S.A.

PFJ?XRCODK,

We have constructed a recombinant DNA library of the measles genome and identified clones containing sequences coding for portions of each of the six viral structural proteins by hybrid-selection/in vitro translation of the viral mRNAs and Northern blotting.-Correlation of the readthrough mRNAs, and of the mRNAs with their in vitro translation products, showed the gene order in measles
virus

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