Separated Transcriptomes of Male Gametophyte and Tapetum in Rice: Validity of a Laser Microdissection (LM) Microarray

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum

The RNA Polymerase PB2 Subunit of Influenza A/HongKong/156/1997 (H5N1) Restrict the Replication of Reassortant Ribonucleoprotein Complexes

BACKGROUND: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. CONCLUSIONS/SIGNIFICANCE: Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes

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

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

The N-terminal fragment of a PB2 subunit from the influenza A virus (A/Hong Kong/156/1997 H5N1) effectively inhibits RNP activity and viral replication.

Influenza A virus has a RNA-dependent RNA polymerase (RdRp) that is composed of three subunits (PB1, PB2 and PA subunit), which assemble with nucleoproteins (NP) and a viral RNA (vRNA) to form a RNP complex in the host nucleus. Recently, we demonstrated that the combination of influenza ribonucleoprotein (RNP) components is important for both its assembly and activity. Therefore, we questioned whether the inhibition of the RNP combination via an incompatible component in the RNP complex could become a methodology for an anti-influenza drug.We found that a H5N1 PB2 subunit efficiently inhibits H1N1 RNP assembly and activity. Moreover, we determined the domains and important amino acids on the N-terminus of the PB2 subunit that are required for a strong inhibitory effect. The NP binding site of the PB2 subunit is important for the inhibition of RNP activity by another strain. A plaque assay also confirmed that a fragment of the PB2 subunit could inhibit viral replication.Our results suggest that the N-terminal fragment of a PB2 subunit becomes an inhibitor that targets influenza RNP activity that is different from that targeted by current drugs such as M2 and NA inhibitors

Promoter binding activity and replication initiation activity of HK PB2 mutants.

<p>(<b>A</b>) Partially purified polymerases analyzed by silver-stained 7.5% SDS-PAGE. Hybrid WSN (W) polymerase replaced only PB2 subunit with HK PB2 wild type (H) (lanes 2), HK PB2 mutants (lanes 3–6) and VN PB2 (V) (lane 7) was transiently expressed in 293T cells and partially purified by using TAP-tagged PA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032634#s2" target="_blank">materials and methods</a>). The numbers in HK PB2 mutants indicate mutated positions in HK PB2. The highlighted numbers are important for the rescue of the RNP activity. (<b>B</b>) UV cross-linking of model vRNA and cRNA promoters to hybrid polymerases. Purified and quantified polymerases were incubated with ³²P-labelled 3′ strand of the vRNA promoter in the presence of the unlabelled 5′ strand of the vRNA promoter (upper panel), or ³²P -labelled 3′ strand of the cRNA promoter in the presence of the unlabelled 5′ strand of the cRNA promoter (lower panel). Lane numbers correspond to the numbers in (A). The positions of the cross-linked products are indicated on the right. (<b>C</b>) ApG synthesis with a model vRNA promoter (upper panel) or cRNA promoter (lower panel). Lane numbers correspond to the numbers in (A). The position of the specific ApG product is indicated on the right. (<b>D and E</b>) Quantification of results obtained in panels B and C, respectively, by phosphorimaging. Data are expressed as percentages relative to VN PB2 (lane 7) (mean ± standard deviation; n = 3). Black bars, model vRNA promoter; white bars, model cRNA promoter. * shows statistical significance at P<0.01 in a Student's t-test.</p

Comparison of RNP activities under thermal stress.

<p>(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.</p

Alignment of PB2 subunit.

<p>(<b>A</b>) Functional map of PB2 subunit. (B) Alignment of amino acid residues in PB2 which are important for the accumulation of RNP. * shows influenza strains used in this study. Gray shading indicates high evolutionary conservation between influenza A, B and C virus sequences.</p

Effect of PA subunit under various thermal stresses.

<p>(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.</p

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

<p>(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.</p