Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs

Abstract

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.

Biology of Influenza A Virus

“…182,184,206 In contrast, HA, NA, and M1 mRNAs are preferentially expressed. 182,184,206 In general, most of the capped and polyadenylated viral mRNAs would be transported from the nucleus to the cytoplasm for protein synthesis. On the other hand, the membrane-bounded proteins, such as HA, NA, and M2, would pass the secretory pathway at the trans-Golgi network for protein maturation.…”

“…183 In addition, cRNA is not polyadenylated. Although both cellular and viral factors have been suggested to have crucial roles in the transcription-replication regulation, 99,184,204,205 the mechanism for the switching from transcription to replication is still poorly understood. Recently, Vreede et al 101 demonstrated that there may be no switch regulating the initiation of RNA synthesis and proposed a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP proteins.…”

“…In general, the amount of viral proteins synthesized in infected cells is largely dependent on the amount of the corresponding mRNA in infected cells. 182,184 Immediately after infection, primary transcription occurs. 182 In this stage, all eight mRNAs are synthesized in equimolar amounts.…”

“…As a consequence, NS1 and NP are the predominant viral proteins in infected cells at this stage. 182,184,206 The reason, however, for the preferential early expression of the NS1 and NP proteins is not known. It is possible that NP is required for the replication and transcription of viral RNA.…”

Chapter 1 Viruses as Model Systems in Cell Biology

“…Influenza virus will infect a number of mammalian cell lines such as MDBK, baby hamster kidney (BHK-21, CCL #lo; ATCC), MDCK, or monkey kidney (African Green monkey kidney, CV-1, CCL #70; ATCC). The time course of viral polypeptide synthesis depends on the strain of virus and the host cell, as well as on the multiplicity of infection (Skehel, 1972;Meier-Ewert and Compans, 1974;Lamb and Choppin, 1976;Shapiro et al, 1987). Thus, it is important to establish the growth properties and time course of replication of a given influenza virus strain in the cell type of interest before planning specific experiments.…”