The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA3cRNA3vRNA), but its transcriptional activity (vRNA3mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNAbinding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.Influenza A virus is a negative-strand RNA virus containing eight segments of single-stranded RNA as its genome (39). The RNA genome is transcribed and replicated by the viral RNA-dependent RNA polymerase in the cell nucleus (21). The viral RNAs (vRNA) are transcribed into mRNAs and replicated through a cRNA intermediate to produce more vRNA molecules. Synthesis of these three RNA species requires different modes of initiation and termination (reviewed in references 23 and 34). Synthesis of mRNAs is primed by short capped RNA fragments that are generated from cellular pre-mRNAs by endonucleolytic cleavage. Consequently, viral mRNA molecules contain a 9-to 17-nucleotide (nt) capped host-derived RNA sequence at their 5Ј ends. On the other hand, the synthesis of cRNA and vRNA molecules is initiated in a primer-independent manner, resulting in triphosphorylated 5Ј ends. Synthesis of mRNAs is prematurely terminated 16 to 17 nucleotides from the 5Ј end of the vRNA template at a sequence of 5 to 7 uridines that acts as a polyadenylation signal (30,47,49). The poly(A) tail is synthesized by the viral RNA polymerase by repeated copying of the U sequence (47). During the synthesis of cRNA molecules, the polyadenylation signal is ignored, resulting in full-length copies of vRNA.All three reactions, i.e., vRNA3mRNA (transcription), vRNA3cRNA (first step of replication), and cRNA3vRNA (second step of replication) are catalyzed b...

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5 and 3 ends and (ii) base pairing in the stems of both the 5 and 3 hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3 hairpin loop were crucial for promoter activity in vivo. However, the 3 hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3 hairpin loop remains unknown.

An R638A mutation of the polymerase acidic protein (PA) subunit of the RNA polymerase of influenza A/WSN/33 virus results in severe attenuation of viral growth in cell culture by promoting the synthesis of defective interfering RNAs. We propose that R638A is an "elongation" mutant that destabilizes PA-RNA template interactions during elongation. A C453R mutation in PA can compensate for this defect, suggesting that amino acids C453 and R638 form part of the same domain.The RNA-dependent RNA polymerase complex of influenza A virus consists of three subunits, polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). All three subunits are required for the transcription (viral RNA [vRNA] 3 mRNA) and replication (vRNA 3 cRNA 3 vRNA) of the eight segments of the negative single-stranded vRNA genome (3). The PB1 subunit forms the core of the complex and is responsible for polymerase activity. The PB2 subunit is involved in generation of capped RNA primers for the initiation of transcription by binding the cap structures of host pre-mRNAs prior to their endonucleolytic cleavage by PB1. The PA subunit is essential for both transcription and replication, but its exact role in the replication cycle of the virus is not clear. Recently, we reported that a histidine-to-alanine mutation at position 510 of PA (H510A) inhibited the endonucleolytic cleavage of capped RNAs, suggesting that PA might be directly involved in the generation of capped primers (4). PA can also induce a generalized proteolysis of both viral and host proteins, but the significance of this observation remains to be determined (9,13,17,19).In a recent report (4), a recombinant A/WSN/33 virus with an R638A mutation in the PA protein that produced pinheadsized plaques on MDBK cells was described. The R638A PA protein was expressed at levels similar to those of the wild-type PA in transfected cells, suggesting that neither the stability nor the proteolytic activity, believed to be associated with PA, was significantly affected. In functional assays, a recombinant polymerase with the R638A mutation in its PA subunit was able to transcribe and replicate a vRNA-like chloramphenicol acetyltransferase (CAT) RNA as determined by measuring CAT activity and RNA levels in transfected cells. Intriguingly, in this transient assay system, the R638A mutant polymerase apparently synthesized increased amounts of CAT vRNA (about a fivefold increase compared to the wild type) but reduced amounts of CAT mRNA (about a fivefold decrease compared to the wild type). In the context of viral infection, however, we did not observe a significant difference in the ratios of vRNA, cRNA, and mRNA for PA, HA, or NA in a primer extension assay (results not shown), suggesting that this discrepancy might be due to the artificial nature of the transient assay used previously. Further studies, employing in vitro tests to assay the promoter binding, endonuclease, and transcription initiation (with ApG or capped RNA) activities of the R638A recombin...

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

In the order Kinetoplastida, genetic exchange has been demonstrated only in the genus Trypanosoma. Analysis of kinetoplast DNA (kDNA) in genetic crosses of T. brucei has shown that whereas maxicircles are inherited uniparentally, minicircles are inherited from both parents. This result was confirmed for a new cross of T. b. brucei and T. b. rhodesiense by restriction enzyme digestion and Southern analysis of purified kDNA. By hybridisation with small minicircle-derived probes, we could demonstrate the presence of particular parental minicircles in the kDNA of hybrid progeny clones. All hybrid clones had inherited two minicircles from one parent despite two of the four clones having maxicircles from the other parent. The results suggest that rather than small-scale exchange of minicircles between parental networks, gross breakdown and reassembly of the minicircle network occurs during genetic exchange.

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The ability of drugs to show therapeutic activity in vitro can be affected by their hydrophobicity (log P). We recently reported the presence of antiparasitic activity within a group of glutathione diesters [I], against Trypanosoma brucei, Leishmania donovanz and Trypanosoma muzi. In this study we show the dependence of antiparasitic activity (ED50) vs log P and indicate an optimum value for the effectiveness of peptidyl drugs against strains of Tb. brucei. Whilst hydrophobicity is a principal component for activity, steric factors also contribute to the observed pattern with these prodrugs. The mechanism of anti-parasitic action of these glutathione prodrugs will be presented and the protein targets qualitatively identified by sensors studies [2].