The NS1 protein of influenza A virus binds not only to poly(A) and a stem-bulge region in U6 small nuclear RNA (snRNA), but also to double-stranded (ds) RNA. Binding assays with NS1 protein mutants established that the previously identified RNA-binding domain of the NS1 protein is required for binding to ds RNA as well as for binding to poly(A) and U6 snRNA. In addition, dsRNA competed with U6 snRNA for binding to the NS1 protein, consistent with both RNAs sharing the same binding site on the protein. As a consequence of its binding to dsRNA, the NS1 protein blocks the activation of the dsRNA-activated protein kinase (PKR) in vitro. This kinase phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (elF-2 alpha), leading to a decrease in the rate of initiation of translation. Assays using purified PKR and purified elF2 demonstrated that the NS1 protein blocks the dsRNA activation of PKR, and experiments using reticulocyte extracts showed that the NS1 protein blocks the inhibition of translation caused by dsRNA activation of PKR. The implications of these results for control mechanisms occurring in influenza virus-infected cells are discussed.

We have shown previously that the influenza virus NS1 protein inhibits the nuclear export of mRNAs. Here we demonstrate that the NS1 protein also regulates another post-transcriptional step: It inhibits pre-mRNA splicing both in vivo and in vitro. The mode by which the NS1 protein inhibits pre-mRNA splicing is novel. The pre-mRNA forms spliceosomes, but subsequent catalytic steps in splicing are inhibited. Affinity selection experiments establish that the NS1 protein is associated with the spliceosomes that are formed. The RNA-binding domain of the NSI protein is required for the inhibition of splicing and for the interaction of the protein with spliceosomes. Because the NS1 protein is associated with U6 snRNA in influenza virus-infected cells as well as in splicing extracts from uninfected cells, it is likely that the NS1 protein also inhibits pre-mRNA splicing in infected cells. Surprisingly, the splicing of the viral nsl mRNA, the very mRNA that encodes the NS1 protein, was resistant to inhibition by the NS1 protein. This resistance is conferred by sequences in nsl mRNA.[Key Words: Influenza virus NS1 protein; pre-mRNA splicing; spliceosome; U6 snRNA] Received May 12, 1994; revised version accepted June 16, 1994.Eukaryotic pre-mRNAs synthesized by nuclear RNA polymerase II undergo a series of post-transcriptional processes. Two of these processes are pre-mRNA splicing and mRNA export from the nucleus, both of which can be regulated (Green 1989;Krug 1993). Alternative splicing, whereby different mRNAs are generated from a single pre-mRNA, has been shown to be regulated by proteins that bind to specific regions of the pre-mRNA precursor. In many instances it has been shown that specific proteins act on specific pre-mRNA targets to regulate their alternative splicing. For example, in the Drosophila sex determination pathway, a specific protein(s)--sex lethal protein or the complex of the Tra and Tra2 proteins--binds to a specific site on a particular premRNA to either suppress or activate, respectively, the usage of a 3' splice site (Baker 1989;Inoue et al. 1990;Bell et al. 1991;Tian and Maniatis 1992).The nuclear export of unspliced pre-mRNAs and spliced mRNAs can also be regulated by specific proteins. Usually unspliced pre-mRNAs are not exported from the nucleus, apparently at least in part because the pre-mRNAs are efficiently committed to spliceosome formation in the nucleus (Legrain and Rosbash 1989). However, this process is overcome in the case of the pre-mRNAs of human immunodeficiency virus-1 (HIV-1) and other lentiviruses by the action of the virus-encoded Rev (or Rev-like) protein, which binds to a specific RNA sequence (RRE, or Rev responsive element) in the viral pre-mRNAs and facilitates their nuclear export (Hadzopoulou-Cladaras et al. 1989; Hanly et al. 1989;Malim et al. 1989). The influenza virus NS1 protein has the opposite effect on the nuclear export of mRNA: It inhibits the transport of mRNAs (Alonso-Caplen et al. 1992;Fortes et al. 1994). The NS1 protein binds to the poly(A) sequence at t...

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Ticks are important vectors of various pathogens that cause infectious diseases in humans. Endosymbiotic bacteria have been explored as targets for tick and tick-borne disease control. However, the tick bacterial community on Hainan Island, which is the largest tropical island in China and has an environment favourable to ticks, has not yet been studied. In this study, we surveyed the bacterial community of ticks collected from grass in one village in Haikou. A total of 20 ticks were morphologically and molecularly identified as Haemaphysalis spp. The tick bacterial 16S rRNA hypervariable region amplicon libraries were sequenced on an Illumina MiSeq platform. A total of 10 possible bacterial genera were detected, indicating a low-diversity bacterial community profile. The dominant bacterial genus, Massilia, accounted for 97.85% of the population. Some other bacterial genera, including Arsenophonus and Pseudomonas, have been reported to play a role in tick development and tick-borne pathogen transmission in other tick species.Overall, the study highlights the first descriptive understanding of the tick bacterial community on Hainan Island and provides a basis for deciphering the interactions between the tick microbiome and tick-borne pathogens.