Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy

Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.Influenza A viruses can infect a wide range of avian and mammalian species (49). Most avian strains of influenza virus infect wild waterfowl and domestic poultry but usually do not spread to humans. However, adaptation of pathogenic avian viruses to humans can occur either by mutation or reassortment, leading to potentially very serious pandemics, as was the case in 1918 when the “Spanish” flu caused 20 to 40 million deaths worldwide (33). Due to this ability to cross the species barrier, influenza A viruses are a permanent threat to human health. Since 2005 the spread of highly pathogenic H5N1 avian strains in Asia, Europe, and Africa has raised serious concern about the potential of this strain to cause an influenza pandemic (50). Since early 2009, an ongoing new, rapidly evolving pandemic threat has arisen from the emergence of a highly contagious, interhuman-transmissible “quadruple reassortant” swine H1N1 virus to which the world population is antigenically naïve (6).Influenza A viruses are enveloped viruses of the orthomyxovirus family whose genomes comprise eight negative-strand RNA segments (2). In contrast to many RNA viruses, the influenza virus genome is transcribed and replicated by the trimeric viral RNA polymerase (PA, PB1, and PB2) in the nuclei of the infected cells. Therefore, the polymerase subunits, which are produced in the cytoplasm, have to be imported into the nucleus and assembled into a functional trimer (2, 18). Many studies have demonstrated that the viral polymerase plays a major role in host specificity, probably due to the necessity for the polymerase subunits to adapt to host cell-interacting partners such as nuclear import factors (13, 16, 25, 37, 46). Due to the lack of in vivo data concerning the interactions between the polymerase subunits in the nucleus and the cytoplasm of the host cells, the mechanisms of polymerase assembly and nuclear import, as well as their spatial and temporal relationships, are still not completely understood. Putative nuclear localization signals (NLSs) have been identified on PB1 (31), PB2 (29), and PA (32), suggesting that each subunit could be imported separately. However, based on in vitro assembly observations and cellular localization studies (8, 9, 12), it has been proposed that PB1 and PA are imported into the nucleus as a subcomplex by import factor RanBP5 (a member of the importin β superfamily). PB2 is thought to enter the nucleus separately, probably via the canonical importin α/importin β pathway (46), and then associates with the PB1/PA heterodimer in the nucleus to form the functional trimeric polymerase. Nevertheless, alternative pathways have also been proposed. Naito et al. (30) suggested that the nuclear import of PB1 requires the formation of a PB2/PB1 heterodimer, stabilized by Hsp90, in the cytoplasm, while PA is transported in the nucleus separately. More recently, a pathway in which the PA/PB2 heterodimer would be formed in the cytoplasm and then imported into the nucleus has been proposed (17). It has also been recently shown that efficient assembly of the trimeric polymerase could be a major limiting factor in the viability of reassortant influenza viruses (26). Since gene reassortment is an evolutionary mechanism of influenza virus which can lead to new strains with pandemic potential, a precise understanding of the processes leading to the formation of an active viral polymerase in the nuclei of infected cells is of great importance.Recent publications have demonstrated that fluorescence cross-correlation spectroscopy (FCCS) is a method of choice to study protein-protein interactions in vivo (23, 27, 42). FCCS is the dual-color extension of fluorescence correlation spectroscopy (FCS), a technique based on the analysis of the temporal fluorescence fluctuations arising from single fluorescently labeled molecules diffusing in and out of the femtoliter-scale detection volume commonly obtained with a confocal microscope. From the autocorrelation of the fluctuating signal, it is possible to extract the local concentrations and mobilities of the molecules of interest (10, 28, 39). In the case of FCCS, signals from two spectrally separated dyes labeling two different molecules are recorded. If the two molecules interact with each other, they diffuse synchronously through the detection volume, resulting in correlated fluctuations in the fluorescence signals acquired in the two channels. The cross-correlation between the two signals is then a direct and quantitative readout of the interactions between the molecular species studied (22, 38, 40). To our knowledge, this study is the first application of FCCS to viral protein interactions and thus provides a general methodological framework to analyze the effects of different host range mutations and the interactions of viral proteins and host factors in the future.In this study, we applied FCCS to monitor the interactions between the subunits of influenza A virus RNA polymerase in live cells. Based both on the study of these interactions in the cytoplasm and nucleus and on the quantitative analysis of the intracellular localization of the subunits, we show that PB1 and PA form a heterodimer in the cytoplasm while PB2 remains a monomer in this compartment. Association of PB1/PA with PB2 to form the trimeric polymerase was detected only in the nucleus, arguing that the PB1/PA heterodimer is normally imported separately from PB2. Interestingly, when we impaired the nuclear import of PB2 by mutating its nuclear localization signal, this induced the aberrant presence of the trimeric polymerase in the cytoplasm and led to the retention of PB1 and PA outside the nucleus. Finally, by comparing the molecular brightnesses of the single polymerase subunits with that of the trimeric complex, we show that trimeric polymerase complexes can interact with each other in the nucleus to form higher-order oligomers.