Artificial hybrids of influenza A virus RNA polymerase reveal PA subunit modulates its thermal sensitivity

Abstract

Background: Influenza A virus can infect a variety of different hosts and therefore has to adapt to different host temperatures for its efficient viral replication. Influenza virus codes for an RNA polymerase of 3 subunits: PB1, PB2 and PA. It is well known that the PB2 subunit is involved in temperature sensitivity, such as cold adaptation. On the other hand the role of the PA subunit in thermal sensitivity is still poorly understood.

Methodology/principal findings: To test which polymerase subunit(s) were involved in thermal stress we reconstituted artificial hybrids of influenza RNA polymerase in ribonucleoprotein (RNP) complexes and measured steady-state levels of mRNA, cRNA and vRNA at different temperatures. The PA subunit was involved in modulating RNP activity under thermal stress. Residue 114 of the PA subunit was an important determinant of this activity.

Conclusions/significance: These findings suggested that influenza A virus may acquire an RNA polymerase adapted to different body temperatures of the host by reassortment of the RNA polymerase genes.

Figure 1. Comparison of RNP activities under thermal stress.

(A) Brief protocol and incubation periods are indicated. (B) Representative analyzed polyacrylamide gel (6%) is shown. * represents statistical significance at p<0.05 in a Student's t-test (n = 3). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were additionally incubated at 34, 37 and 42°C for 9 hours, respectively. Then total RNAs were extracted and analyzed by primer extension assay. A/WSN/33, A/Hong Kong/156/97, A/NT/60/68, A/Vietnam/1194/2004 and pandemic H1N1 2009 virus are abbreviated as WSN, HK, NT, VN and SW, respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively.

Figure 2. Time course of RNP activity on thermal stress at 42°C.

(A) Brief protocol and incubation periods are indicated. (B) Quantitation of cRNA (closed circle) and vRNA (opened circle) of WSN at 42°C is shown. Data have been normalized for total RNA using the 5S rRNA signal. Data are expressed as a percentage of wild type activity (with standard deviation). Quantitation was based on at lease three independent sets of data. (C) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Then pre-incubated cells were transferred to 42°C. Total RNAs were extracted and analyzed at each time point by primer extension assay. (D) RNA polymerase subunits (PA, PB1 and PB2) and NP - transiently expressed in human 293T cells, analyzed by western blotting using specific anti-bodies of each subunit by 8% SDS-PAGE. A/WSN/33, A/HongKong/156/97 and pandemic H1N1 2009 virus are abbreviated as WSN, HK and SW, respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively.

Figure 3. Effect of transient heat shock at 42°C on RNP activity.

(A) Brief protocol and incubation periods are indicated. (B) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were stimulated by 42°C for 15 min, and then continued to incubate at 37°C up to 6 hours. Total RNAs were extracted and analyzed by primer extension assay. A/WSN/33 and A/HongKong/156/97 are abbreviated as WSN and HK, respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively.

Figure 4. Analysis of the contribution of the RNA polymerase subunit(s) to thermal stress.

(A) Representative analyzed polyacrylamide gel (6%) is shown. WSN (W) subunit(s) was replaced with corresponding VN (V) subunit(s). 293T cells expressing the hybrid RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were additionally incubated at 37°C or 42°C for 9 hours. Total RNAs were extracted and analyzed by primer extension assay. Each RNA polymerase subunit is indicated as PB1, PB2 or PA. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively. (B) Quantitation of cRNA (closed bar) and vRNA (opened bar) and standard deviations of bands in panel A expressed as a percentage of WSN strain at 37°C. ** represents statistical significance at p<0.01 in a Student's t-test (n = 3). Each RNA polymerase subunit is indicated as PB1, PB2 or PA.

Figure 5. Effect of PA subunit under various thermal stresses.

(A) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). WSN PA subunit was replaced with that of each strain. 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were additionally incubated at 34°C, 37°C or 42°C for 9 hours. Total RNAs were extracted and analyzed by primer extension assay. A/WSN/33, A/HongKong/156/97, A/NT/60/68, A/Vietnam/1194/2004 and pandemic H1N1 2009 virus are abbreviated as W, H, N, V and S respectively. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively. (B) Quantitation of cRNA (closed bar) and vRNA (opened bar) and standard deviations of bands in panel A expressed as a percentage of WSN strain at 37°C. ** represents statistical significance at p<0.01 in a Student's t-test (n = 3). Each RNA polymerase subunit is indicated as PB1, PB2 or PA.

Figure 6. Mutagenesis of the PA subunit and thermal stress.

(A) Alignment of PA subunits. Interesting amino acids of WSN PA subunit are indicated. (B) Representative analyzed polyacrylamide gel (6%) is shown (n = 3). WSN PA subunit was mutated at position 86, 114 or 556 as indicated in (A). 293T cells expressing influenza RNP were incubated at 37°C for 24 hours as pre-incubation. Pre-incubated cells were additionally incubated for 9 hours under thermal stress (42°C). Total RNAs were extracted and analyzed by primer extension assay. 5s ribosomal RNA (rRNA) is indicated as an internal control. mRNA, cRNA and vRNA are viral messenger RNA, complementary viral RNA and viral RNA, respectively. (C) The each RNP activity is graphically visualized. Wild type of PA subunit is indicated as WSN (WT). The activity is expressed as a % relative to the WSN wild-type. White, light oblique and dark oblique lined columns show steady-state levels of mRNA, cRNA and vRNA, respectively. * and ** show statistically significant differences from WSN wild-type at p<0.05 and p<0.01 in a Student's t-test.