Interferon induction and/or production and its suppression by influenza A viruses

Abstract

Developmentally aged chicken embryo cells which hyperproduce interferon (IFN) when induced were used to quantify IFN production and its suppression by eight strains of type A influenza viruses (AIV). Over 90% of the IFN-inducing or IFN induction-suppressing activity of AIV populations resided in noninfectious particles. The IFN-inducer moiety of AIV appears to preexist in, or be generated by, virions termed IFN-inducing particles (IFP) and was detectable under conditions in which a single molecule of double-stranded RNA introduced into a cell via endocytosis induced IFN, whereas single-stranded RNA did not. Some AIV strains suppressed IFN production, an activity that resided in a noninfectious virion termed an IFN induction-suppressing particle (ISP). The ISP phenotype was dominant over the IFP phenotype. Strains of AIV varied 100-fold in their capacity to induce IFN. AIV genetically compromised in NS1 expression induced about 20 times more IFN than NS1-competent parental strains. UV irradiation further enhanced the IFN-inducing capacity of AIV up to 100-fold, converting ISP into IFP and IFP into more efficient IFP. AIV is known to prevent IFN induction and/or production by expressing NS1 from a small UV target (gene NS). Evidence is presented for an additional downregulator of IFN production, identified as a large UV target postulated to consist of AIV polymerase genes PB1 + PB2 + PA, through the ensuing action of their cap-snatching endonuclease on pre-IFN-mRNA. The products of both the small and large UV targets act in concert to regulate IFN induction and/or production. Knowledge of the IFP/ISP phenotype may be useful in the development of attenuated AIV strains that maximally induce cytokines favorable to the immune response.

FIG. 1.

Time course of IFN production at 40.5°C from developmentally aged primary CEC induced with influenza virus strains: (A) A/TK/WI/66; (B) parental A/TK/OR/71 or A/TK/OR/71(delNS1[1-124]); (C) parental A/PR/8/34 or A/PR/8/34(ΔNS1). The multiplicity used at time zero characteristically induced maximal yields of IFN for each strain. Panel A is representative of three other strains (not shown) which displayed peak yields of IFN at about 24 h postinduction. Note the change in scale for each ordinate.

FIG. 2.

IFN induction dose (multiplicity)-response (IFN yield) curves generated by AIV in aged primary CEC at 40.5°C. The growth medium was harvested 24 h postinduction, processed, and assayed for IFN: (A) parental A/TK/OR/71 and A/TK/OR/71(delNS1[1-124]); (B) parental A/PR/8/34 and A/PR/8/34(ΔNS1). Note changes in scale for each abscissa. A comparable curve representative of the other four strains used in this study is shown in Fig. 4.

FIG. 3.

Histogram showing the maximum yields of IFN produced following optimal conditions of induction in aged primary CEC at 40.5°C for each of the eight strains of influenza virus tested. IFN yields are averaged from two to four independent determinations.

FIG. 4.

Determination of IFP in a population of egg-derived influenza virus. Monolayers containing 10⁷ aged primary CEC were infected with various amounts of A/TK/WI/66 and incubated at 40.5°C for 24 h, and then the medium was assayed for IFN. Based on a Poisson distribution of virus among the cell population, the dilution of virus that induces 0.63 of the maximum yield of virus contains on average 1 IFP (; see text).

FIG. 5.

Determination of ISP in an influenza virus-infected population. Monolayers containing 10⁷ aged primary CEC were infected simultaneously with (i) UV-A/TK/WI/66 at a multiplicity of IFP that infected all cells and produced maximal yields of IFN and (ii) increasing multiplicities of A/TK/OR/71 to assess its ISP activity. Cells infected only with IFP induced 105,000 U/10⁷ cells in 24 h (i.e., 1.0 of maximum IFN yield [upper dashed line]), whereas those infected only with the putative ISP produced maximally 600 U/10⁷ cells (lower dashed line). The solid line represents the yield of IFN as a function of increasing multiplicity of ISP. Based on a Poisson distribution of ISP in the cell monolayer, the fraction of cells producing 0.37 of the maximum IFN yield is assumed to contain on average 1 ISP (52). See text for calculation of ISP and the ISP/IP ratio.

FIG. 6.

IFN-inducing capacity of UV-irradiated influenza viruses. Stock preparations of virus were diluted 1:2 in attachment solution, exposed to UV (254 nm) radiation for various periods of time, and then used to induce IFN on aged primary CEC. All cells were infected with the same amount of virus, which when unirradiated induced maximal yields of IFN. This is represented by the IFN yield shown at time zero. Both the time of exposure to UV radiation and the effective dose are displayed on the lower abscissas. The number of lethal UV hits delivered to the entire genome is displayed on the upper abscissa, where 1 lethal UV hit = 82.8 ergs/mm². The time of exposure to UV radiation (seconds) and the dose (in ergs per square millimeter) are shown on the lower abscissas. The IFN yield is plotted as a function of UV dose for each of the eight strains examined. (A) A/TK/WI/66 and A/WSN/33; (B) A/TK/ONT/7732/66 and A/TK/ONT/7732/66(Clone 1B); (C) A/TK/OR/71 and A/TK/OR/71(delNS1[1-124]); (D) A/PR/8/34 and A/PR/8/33(ΔNS1).

FIG. 7.

Comparison of the maximum yields of IFN induced by active and UV-irradiated AIV. Shown are the maximum yields of IFN induced by eight AIV strains following infection of aged CEC by egg-derived stocks of virus, either active (black bars) or UV irradiated (gray bars). The number of lethal UV hits required to achieve maximum IFN-inducing capacity is displayed over each bar.

FIG. 8.

Comparison of the maximum yields of IFN induced by parental virus and their genetically NS1-compromised counterparts before and after UV irradiation. The number of lethal UV hits required to achieve maximum IFN-inducing capacity is displayed over each bar.

FIG. 9.

Survival of PFP activity and IFN-inducing capacity of A/TK/OR/71 as a function of time at 50.0°C. Aliquots of virus removed from the heat at the times indicated were cooled immediately and tested for both the fraction of surviving PFP activity and the level of IFN induced by a multiplicity of virus which produced maximal yields of IFN from active virus. Viral hemagglutinating activity was not affected by the longest period of exposure to heat. The results are presented as the fraction of surviving activity of PFP and IFN yield relative to those in unheated samples. The IFN-inducing capacity of the virus before heating was 600 U/10⁷ cells. Each point represents the average of three assays in two tests.

FIG. 10.

Comparison of the fraction of maximum IFN yield observed (data points and solid line) with the expected fraction of inactivation of four potential UV targets based on a Poisson distribution of UV hits to an AIV population as a function of UV dose (dashed lines). A/TK/OR/71(delNS1[1-124]) was exposed to different doses of UV radiation, and its capacity to induce IFN was assessed by infecting aged CEC with the same amount of virus shown to induce maximal amounts of IFN in its active state: in the example illustrated, ≈8,000 U/10⁷cells. The data points represent two independent determinations where the peak yields of IFN were 165,000 and 130,000 U/10⁷ cells. The dashed lines represent the curves expected for the fraction of the AIV population inactivated for the following gene(s): (A) NS; (B) PB1 = PB2 ≈ PA; (C) any combination of two polymerase genes; (D) PB1 + PB2 + PA. The genes for HA, NP, NA, and M are not shown but would fall between the NS gene (A) and any of the polymerase genes acting singly (B). The sizes of the gene segments were taken from Lamb and Krug (35) to calculate the rate of inactivation based on the D₃₇ for each gene or collection of genes in the virus population relative to the inactivation of infectivity, where D₃₇ = 82.8 ergs/mm².