The N-terminal fragment of PA subunit of the influenza A virus effectively inhibits ribonucleoprotein (RNP) activity via suppression of its RNP expression.

The influenza RNP, which is formed from PB1, PB2, PA, NP subunits, and vRNA, is autonomously replicated and transcribed in the infected cell. The simplest method to inhibit RNP activity is to impair the formation of the RNP. Thereupon we confirmed whether the peptides/fragments mimicking one of RNP components can interfere with their formation. During the process of this inhibitory study we found interesting suppression of protein expression of the RNP components by the N-terminal fragment of PA subunit. Especially, we found two residues (D108 and K134) on the fragment that were critical for the suppression. Furthermore, we determined the combination of three amino acids (P28, M86 and E100) on the fragment that are important for the strong suppression, and identified the minimum essential region (residues from 1 to 188) of the PA subunit that allowed its suppression. Our findings indicate that the N-terminal fragment of PA subunit may become one of candidates for an effective inhibitor of influenza RNP activity.

Co-incorporation of the PB2 and PA polymerase subunits from human H3N2 influenza virus is a critical determinant of the replication of reassortant ribonucleoprotein complexes.

The influenza virus RNA polymerase, composed of the PB1, PB2 and PA subunits, has a potential role in influencing genetic reassortment. Recent studies on the reassortment of human H3N2 strains suggest that the co-incorporation of PB2 and PA from the same H3N2 strain appears to be important for efficient virus replication; however, the underlying mechanism remains unclear. Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes and demonstrated that the RNP activity was severely impaired when the PA subunit of H3N2 strain A/NT/60/1968 (NT PA) was introduced into H1N1 or H5N1 polymerase. The NT PA did not affect the correct assembly of the polymerase trimeric complex, but it significantly reduced replication-initiation activity when provided with a vRNA promoter and severely impaired the accumulation of RNP, which led to the loss of RNP activity. Mutational analysis demonstrated that PA residues 184N and 383N were the major determinants of the inhibitory effect of NT PA and 184N/383N sequences were unique to human H3N2 strains. Significantly, NT PB2 specifically relieved the inhibitory effect of NT PA, and the PB2 residue 627K played a key role. Our results suggest that PB2 from the same H3N2 strain might be required for overcoming the inhibitory effect of H3N2 PA in the genetic reassortment of influenza virus.

Intrahost emergent dynamics of oseltamivir-resistant virus of pandemic influenza A (H1N1) 2009 in a fatally immunocompromised patient.

The oseltamivir-resistant pandemic influenza virus A (2009 H1N1) with H275Y mutation in neuraminidase (NA) has been sporadically reported, and its wide spread remains a potential threat. Here we detected the uneven distribution of H275Y mutant virus in a patient who received a 21-day long-term administration of oseltamivir. Intrahost variation of the virus showed that the H275Y mutant virus was the predominant population in both nasopharynx and right lung, whereas the oseltamivir-sensitive virus comprised half the population in the left lung. By constructing minimum spanning trees, it is proposed that the H275Y mutant might be generated primarily in the nasopharynx, then spread to the right and left lungs.

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.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

New phenolic compounds from Camellia sinensis L. fermented leaves.

Two new phenolic compounds (1 and 2, named as teasperol and teasperin, respectively) were isolated from fermented tea (Camellia sinensis L.) products, together with known phenolic compounds. Teasperol (1) was isolated from Chinese traditional post-fermented tea leaves (Liu-pao tea) and teasperin (2) was from a Japanese tea product which was selectively fermented with Aspergillus sp. The chemical structures of 1 and 2 were elucidated based on the analyses of their spectroscopic data.

The RNA polymerase PB2 subunit of influenza A/HongKong/156/1997 (H5N1) restricts the replication of reassortant ribonucleoprotein complexes [corrected].

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.

Clinical study concerning the relationship between community-acquired pneumonia and viral infection in northern Thailand.

OBJECTIVE: The etiological agents associated with community-acquired pneumonia (CAP) in Thailand have been studied extensively in bacterial pathogens, but not in viral pathogens. To clarify the association of viral pathogens with CAP, we conducted a comprehensive study of viral and bacterial pathogens in patients with CAP. METHODS: We enrolled 119 hospitalized patients with CAP in Nakornping Hospital, Chiang Mai, Thailand between 2006 and 2008. The severity of pneumonia was classified and the risk factors for death were estimated. Bacterial and fungal pathogens were determined from specimens taken from blood and sputum, and viral pathogens were identified from nasopharyngeal specimens by RT-PCR using primers specific for 7 respiratory viruses. RESULTS: Overall, 29 patients were HIV-infected and 90 patients were non-HIV-infected. The microbial pathogens most commonly isolated among HIV-infected patients were: 4 Klebsiella pneumoniae, 4 Mycobacterium tuberculosis and 3 Haemophilus influenzae. Among non-HIV infected patients, predominant microbial pathogens were: 6 Pseudomonas aeruginosa, 5 Haemophilus influenzae and 4 Klebsiella pneumoniae. As for viral pathogens for CAP, influenza virus was identified from 2 HIV-infected patients and 5 non-HIV infected patients. In addition, human rhinovirus (HRV) and respiratory syncytial virus (RSV) were identified from 2 patients each among non-HIV-infected patients. CONCLUSION: Our study demonstrates that the most common viral agent was influenza virus (5%), followed by HRV (2%) and RSV (2%) among CAP patients in northern Thailand. The underlying chronic obstructive pulmonary disease (COPD) seems to be correlated with the severity of illness.

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.