The first step in the replication of influenza virion RNAs is the synthesis of full-length transcripts of these RNAs. The synthesis of these transcripts, or template RNAs, requires: (i) unprimed initiation rather than the capped RNAprimed initiation used during viral mRNA synthesis, and (it) antitermination at the polyadenylylation site used during mRNA synthesis. To determine the mechanism of template RNA synthesis, we prepared nuclear extracts from infected cells that were active in the synthesis of both template RNAs and viral mRNAs. By providing the dinucleotide ApG as primer, we circumvented the inefficient unprimed initiation catalyzed by these extracts and, as a consequence, were able to focus on the antitermination step. Antitermination, and hence template RNA synthesis, occurred when ApG but not a capped RNA was used as primer, indicating that the presence of a 5' capped end blocked antitermination at the 3' end of the transcript. Ultracentrifugation of the nuclear extract yielded a pellet fraction that contained viral nucleocapsids active in viral mRNA synthesis but not template RNA synthesis and a supernatant fraction that contained the antitermination factor. When the supernatant, which had essentially no activity by itself, was added to the pellet in the presence of ApG, template RNA synthesis was restored. Depletion experiments in which this supernatant was incubated with protein A-Sepharose containing antibodies to individual viral proteins demonstrated that the viral nucleocapsid protein was required for antitermination. The implications of these results for the control of viral RNA replication are discussed.The synthesis of influenza virus mRNA is primed by capped RNA fragments derived from host-cell RNA polymerase II transcripts (1-3). These primers are generated by a viral cap-dependent endonuclease that cleaves RNA polymerase II transcripts 10-13 nucleotides from their 5' ends preferentially at a purine residue. Viral mRNA chains are elongated until a stretch of uridine residues is reached 17-22 nucleotides before the 5' end of virion RNA (vRNA) is reached, where transcription terminates and poly(A) is added to the mRNAs (4, 5). Viral mRNA synthesis is catalyzed by nucleocapsids from purified virions (2), and some of the roles of the constituent nucleocapsid-associated polymerase (P) proteins (PB1, PB2, and PA) have been delineated by previous experiments (6-8).For replication to occur, an alternative type of transcription is required that results in the production of full-length copies of vRNA. These copies can then serve as the templates for new vRNA. The full-length transcripts, or template RNAs, are initiated without a primer and are not terminated at the poly(A) site used during mRNA synthesis (4, 9). In vivo, the switch from viral mRNA to template RNA synthesis requires the synthesis of one or more virus-specific proteins (9, 10).To identify these proteins and to determine their roles in the switch from viral mRNA to template RNA synthesis, it is necessary to establish an in vit...

MAs are powerful tools for differential screening of tears for large numbers of trace proteins. Analysis allowed the identification of previously undetected proteins that may participate in the host defense system as well as demonstrated the profound change in tear composition associated with prolonged eye closure in a manner reflective of physiological function.

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Stationary phase antibody array methodology was useful for the screening of various cytokines, growth factors and MMPs in tears. These analyses identified in tears of VKC patients previously unreported factors including MMP-3 and MMP-10 and multiple proteases, growth factors and cytokines, which may all play an important role in the pathogenesis of conjunctival inflammation.

Influenza viral RNA transcription in vitro is primed by capped RNA fragments cleaved from capped RNAs by a viral endonuclease. The present study was undertaken to determine whether the specificities of the viral endonuclease and transcriptase observed in in vitro studies are also observed in the infected cell. The NS (nonstructural) gene of influenza WSN virus was cloned in pBR322 by using a double-stranded DNA containing a cDNA copy of both virion RNA (vRNA) and in vivo viral mRNA. We determined the 5' terminal sequence of the particular NS viral mRNA molecule which was cloned and also the 5' terminal sequences of the entire population of in vivo NS viral mRNAs synthesized in two different cell lines. For the latter determination we used a restriction fragment from the cloned DNA for the reverse transcriptase-catalyzed extension of total in vivo viral mRNA. The results indicate that in vivo and in vitro viral RNA transcription are similar in two important respects: (i) transcription initiates not with an A residue directed by the 3' terminal U of the vRNA, but with a G residue directed by the 3' penultimate C of the vRNA; and (ii) capped RNA fragments containing a 3' terminal A residue are preferentially used as primers, therapy generating an AG sequence in the viral mRNA complementary to the 3' terminal UC of the vRNA. Actually, for in vivo transcription, a subset of A-terminated capped fragments, namely those containing a 3' penultimate C residue, are the preferred primers. The latter specificity had not been observed in previous in vitro studies.