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The two steps in influenza virus RNA replication are (i) the synthesis of template RNAs, i.e., full-length copies of the virion RNAs, and (ii) the copying of these template RNAs into new virion RNAs. We prepared nuclear extracts from infected HeLa cells that catalyzed both template RNA and virion RNA synthesis in vitro in the absence of an added primer. Antibody depletion experiments implicated nucleocapsid protein molecules not associated with nucleocapsids in template RNA synthesis for antitermination at the polyadenylation site used during viral mRNA synthesis. Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleocapsid protein was indeed required for template RNA synthesis both in vivo and in vitro. Nuclear extracts prepared from mutant virus-infected cells synthesized template RNA at the permissive temperature but not at the nonpermissive temperature, whereas the synthesis of mRNA-size transcripts was not decreased at the nonpermissive temperature. Antibody depletion experiments showed that nucleocapsid protein molecules not associated with nucleocapsids were also required for the copying of template RNA into virion RNA. In contrast to the situation with the synthesis of transcripts complementary to virion RNA, no discrete termination product(s) were made during virion RNA synthesis in vitro in the absence of nucleocapsid protein molecules.

Single-stranded M13 DNAs specific for various influenza virus genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results show that influenza virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs, viral mRNAs, and viral proteins were coupled. Thus, the NS (nonstructural) virion RNA was preferentially synthesized early, leading to the preferential synthesis of NS1 viral mRNA and NS1 protein; in contrast, M (matrix) RNA synthesis was delayed, leading to the delayed synthesis of Ml viral mRNA and Ml protein. This phase lasted for 2.5 h in BHK-21 cells, the time at which the rate of synthesis of all the viral mRNAs was maximal. During the second phase, the synthesis of all the virion RNAs remained at or near maximum until at least 5.5 h postinfection, whereas the rate of synthesis of all the viral mRNAs declined dramatically. By 4.5 h, the rate of synthesis of all the viral mRNAs was 5% of the maximum rate. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed prinicipally by previously synthesized viral mRNAs. Short pulses (3 min) with [3H]uridine and nonaqueous fractionation of cells were used to show that influenza virion RNA synthesis occurred in the nucleus, demonstrating that all virus-specific RNA synthesis was nuclear. Virion RNAs, like viral mRNAs, were efficiently transported to the cytoplasm at both early and late times of infection. In contrast, the full-length transcripts of the virion RNAs, which are the templates for virion RNA synthesis, were sequestered in the nucleus. Thus, the template RNAs, which were synthesized only at early times, remained in the nucleus to direct virion RNA synthesis throughout infection. These results enabled us to present an overall scheme for the control of influenza virus gene expression.

Interferons alpha and beta induce an efficient antiviral state against influenza virus in mouse cells that possess the Mx gene, but not in mouse cells that lack this gene. In Mx-containing cells treated with interferon the amount of viral mRNA synthesized as a result of primary transcription is drastically reduced. Only two viral mRNAs could be detected by Northern analysis and by translating the poly(A)+ RNA from infected cells in wheat germ extracts: a reduced amount of the mRNA for nonstructural protein 1 and an even lower amount of the mRNA for the matrix protein. The other viral mRNAs were not made in detectable amounts. In addition, the rate of viral mRNA synthesis catalyzed by the inoculum transcriptase, measured by in vitro RNA synthesis catalyzed by permeabilized cells, was severely inhibited. In contrast, interferon treatment of cells lacking the Mx gene had little or no effect on either the steady-state level or the rate of synthesis of viral mRNAs made by the inoculum transcriptase. These results indicate that the interferon-induced Mx gene product, a 75,000-molecular-weight protein that accumulates in the nucleus, inhibits influenza viral mRNA synthesis which occurs in the nucleus. No Mx-specific effect acting directly on viral protein synthesis in the cytoplasm was observed.