It has been speculated that before vertebrates evolved somatic diversity-based adaptive immunity, the germline-encoded diversity of innate immunity may have been more developed. Amphioxus occupies the basal position of the chordate phylum and hence is an important reference to the evolution of vertebrate immunity. Here we report the first comprehensive genomic survey of the immune gene repertoire of the amphioxus Branchiostoma floridae. It has been reported that the purple sea urchin has a vastly expanded innate receptor repertoire not previously seen in other species, which includes 222 toll-like receptors (TLRs), 203 NOD/NALP-like receptors (NLRs), and 218 scavenger receptors (SRs). We discovered that the amphioxus genome contains comparable expansion with 71 TLR gene models, 118 NLR models, and 270 SR models. Amphioxus also expands other receptor-like families, including 1215 C-type lectin models, 240 LRR and IGcam-containing models, 1363 other LRR-containing models, 75 C1q-like models, 98 ficolin-like models, and hundreds of models containing complement-related domains. The expansion is not restricted to receptors but is likely to extend to intermediate signal transducers because there are 58 TIR adapter-like models, 36 TRAF models, 44 initiator caspase models, and 541 death-fold domain-containing models in the genome. Amphioxus also has a sophisticated TNF system and a complicated complement system not previously seen in other invertebrates. Besides the increase of gene number, domain combinations of immune proteins are also increased. Altogether, this survey suggests that the amphioxus, a species without vertebrate-type adaptive immunity, holds extraordinary innate complexity and diversity.

Double-stranded (ds) RNA, a common component of virus-infected cells, is a potent inducer of the type I interferon and other cellular genes. For identifying the full repertoire of human dsRNA-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from dsRNA-treated GRE cells. Because these cells lack all type I interferon genes, the possibility of gene induction by autocrine actions of interferon was eliminated. Our screen identified 175 dsRNA-stimulated genes (DSG) and 95 dsRNA-repressed genes. A subset of the DSGs was also induced by different inflammatory cytokines and viruses demonstrating interconnections among disparate signaling pathways. Functionally, the DSGs encode proteins involved in signaling, apoptosis, RNA synthesis, protein synthesis and processing, cell metabolism, transport, and structure. Induction of such a diverse family of genes by dsRNA has major implications in host-virus interactions and in the use of RNA i technology for functional ablation of specific genes.

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.Precise and timely subcellular localization of proteins is essential for their biological functions, and conversely, the control of protein localization provides the cells with a convenient way to regulate their functions. Posttranslational modifications of proteins play critical roles in these aspects. For instance, three of the most important outcomes after protein phosphorylation are typically the changes in protein localization, stability, or activity (32, 38). Eukaryotic DNA replication is strictly controlled by mechanisms that regulate the cell cycle to ensure both genetic inheritance and stability. DNA replication is initiated at a precise time when the cellular replication machinery is ready, and then the genome is replicated for a single round in each cell cycle. Cell cycle entry and progression are regulated by cyclin/cyclin-dependent kinases (CDKs) that phosphorylate key regulatory proteins (5,59,65,74). At the G 1 /Sphase transition, cyclin/CDK complexes phosphorylate the components of the cellular DNA prereplication complex (pre-RC), including origin recognition complex, Cdc6, Cdt1, and MCM2-7, enabling the initiation of replication and subsequently preventing reduplication in the same cell cycle (5, 6, 54). The specific mechanisms vary for different components in different organisms. For example, for the replicative DNA helicase complex composed of the MCM2-7 subunits (71), phosphorylation inhibits its activity in humans and mice (29,30). In budding yeast, phosphorylation leads t...

The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.The papillomavirus E2 protein is a multifunctional nuclear phosphoprotein with a molecular mass of approximately 40 kDa. With high affinity and specificity, it functions as a dimer, binding to multiple copies of a consensus E2 binding site (E2BS), ACCN 6 GGT, located in the upstream regulatory region (URR) and regulating both viral DNA replication and mRNA transcription from the viral E6 promoter located immediately upstream of the E6 gene. The E2 protein is the primary origin recognition protein and helps recruit the viral replication initiator E1 protein (8,30,40,55,64,66,70). It also plays a role in the assembly of the preinitiation complex (39; K.-Y. Lee, T. R. Broker, and L. T. Chow, unpublished data). In vitro, E2 is able to exclude nucleosome formation on the origin, which would otherwise inhibit the initiation of DNA replication (36). Furthermore, E2 modulates the native human papillomavirus (HPV) URR-E6 promoter or a surrogate promoter linked to the URR or to one or more copies of synthetic E2BS (16-18, 26, 59, 62, 63). The bovine papillomavirus type 1 (BPV-1) E2 protein tethers the BPV-1 DNA to mitotic chromosomes, ensuring plasmid segregation during mitosis (28,35,58). Moreover, the BPV-1 E2 has been postulated to play a role in virion morphogenesis (14).The fu...

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