Inhibition of influenza virus replication in cultured cells by RNA-cleaving DNA enzyme

AbstractInfluenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection

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

The N-Terminal Region of the PA Subunit of the RNA Polymerase of Influenza A/HongKong/156/97 (H5N1) Influences Promoter Binding

BACKGROUND: The RNA polymerase of influenza virus is a heterotrimeric complex of PB1, PB2 and PA subunits which cooperate in the transcription and replication of the viral genome. Previous research has shown that the N-terminal region of the PA subunit of influenza A/WSN/33 (H1N1) virus is involved in promoter binding. METHODOLOGY/PRINCIPAL FINDINGS: Here we extend our studies of the influenza RNA polymerase to that of influenza strains A/HongKong/156/97 (H5N1) and A/Vietnam/1194/04 (H5N1). Both H5N1 strains, originally isolated from patients in 1997 and 2004, showed significantly higher polymerase activity compared with two classical human strains, A/WSN/33 (H1N1) and A/NT/60/68 (H3N2) in vitro. This increased polymerase activity correlated with enhanced promoter binding. The N-terminal region of the PA subunit was the major determinant of this enhanced promoter activity. CONCLUSIONS/SIGNIFICANCE: Overall we suggest that the N-terminal region of the PA subunit of two recent H5N1 strains can influence promoter binding and we speculate this may be a factor in their virulence

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

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

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

a<p>The replication (cRNA and vRNA) and transcription (mRNA) activities are indicated as a relative activity (%) ± S.D. from start point (0 hours) (n = 3).</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