Pre-mRNA Processing Factor Prp18 Is a Stimulatory Factor of Influenza Virus RNA Synthesis and Possesses Nucleoprotein Chaperone Activity.

The genome of influenza virus (viral RNA [vRNA]) is associated with the nucleoprotein (NP) and viral RNA-dependent RNA polymerases and forms helical viral ribonucleoprotein (vRNP) complexes. The NP-vRNA complex is the biologically active template for RNA synthesis by the viral polymerase. Previously, we identified human pre-mRNA processing factor 18 (Prp18) as a stimulatory factor for viral RNA synthesis using a Saccharomyces cerevisiae replicon system and a single-gene deletion library of Saccharomyces cerevisiae (T. Naito, Y. Kiyasu, K. Sugiyama, A. Kimura, R. Nakano, A. Matsukage, and K. Nagata, Proc Natl Acad Sci USA, 104:18235-18240, 2007, https://doi.org/10.1073/pnas.0705856104). In infected Prp18 knockdown (KD) cells, the synthesis of vRNA, cRNA, and viral mRNAs was reduced. Prp18 was found to stimulate in vitro viral RNA synthesis through its interaction with NP. Analyses using in vitro RNA synthesis reactions revealed that Prp18 dissociates newly synthesized RNA from the template after the early elongation step to stimulate the elongation reaction. We found that Prp18 functions as a chaperone for NP to facilitate the formation of NP-RNA complexes. Based on these results, it is suggested that Prp18 accelerates influenza virus RNA synthesis as an NP chaperone for the processive elongation reaction. IMPORTANCE: Templates for viral RNA synthesis of negative-stranded RNA viruses are not naked RNA but rather RNA encapsidated by viral nucleocapsid proteins forming vRNP complexes. However, viral basic proteins tend to aggregate under physiological ionic strength without chaperones. We identified the pre-mRNA processing factor Prp18 as a stimulatory factor for influenza virus RNA synthesis. We found that one of the targets of Prp18 is NP. Prp18 facilitates the elongation reaction of viral polymerases by preventing the deleterious annealing of newly synthesized RNA to the template. Prp18 functions as a chaperone for NP to stimulate the formation of NP-RNA complexes. Based on these results, we propose that Prp18 may be required to maintain the structural integrity of vRNP for processive template reading.

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone.

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.

Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins.

The processes governing chromatin remodeling and assembly, which occur prior to and/or after transcription and replication, are not completely understood. To understand the mechanisms of transcription and replication from chromatin templates, we have established in vitro replication and transcription systems using adenovirus (Ad) DNA complexed with viral basic core proteins, called Ad core, as a template. Using this system, we have previously identified, from HeLa cells, template activating factor-I as a stimulatory factor for the Ad core DNA replication. Here, using this system as a tool, we identified and purified a novel template activating factor activity that consists of two acidic polypeptides whose apparent molecular masses are 38 kDa and 37 kDa. These two polypeptides correspond to two splicing variants of nucleolar phosphoprotein, nucleophosmin/B23. Recombinant B23 proteins stimulate the Ad core DNA replication, and the acidic regions of B23 proteins are important for its activity. In addition, B23 proteins directly bind to core histones and transfer them to naked DNA. Furthermore, chromatin components such as histones and topoisomerase II are co-immunoprecipitated with B23 from cell extracts. These observations lead to a hypothesis that nucleophosmin/B23 is involved in structural changes of chromatin, thereby regulating transcription and replication within the ribosomal DNA region or maintaining the nucleolar structure.

Histone- and chromatin-binding activity of template activating factor-I.

Template activating factor-I (TAF-I) is a histone-binding chromatin remodeling factor. We recently found that TAF-I is capable of mediating decondensation of Xenopus sperm chromatin by releasing sperm-specific basic proteins. Here we present evidence that TAF-I preferentially binds to histone H3 among four core histones. Immunofluorescent staining revealed that TAF-I binds to the decondensed sperm chromatin, of which protein components predominantly consist of histones H3 and H4.

Coiled-coil structure-mediated dimerization of template activating factor-I is critical for its chromatin remodeling activity.

Template activating factor-I (TAF-I)alpha and TAF-Ibeta have been identified as the host factors that activate DNA replication of the adenovirus genome complexed with viral basic core proteins (Ad core). TAF-I causes a structural change of the Ad core, thereby stimulating not only replication but also transcription from the Ad core DNA in vitro. TAF-I also activates transcription from the reconstituted chromatin consisting of DNA fragments and purified histones through chromatin remodeling. Although the carboxyl-terminal region, which is highly rich in acidic amino acids, is essential for the TAF-I activity, it remains unclear how other parts are involved in its activity. The native TAF-I isolated from HeLa cells exists as either hetero- or homo-oligomer. Here, we have demonstrated by cross-linking assays that most of TAF-I exists as a dimer. Analyses using deletion mutant TAF-I proteins revealed that the amino-terminal region of TAF-I common to both alpha and beta is essential for dimerization. This region is predicted to form a coiled-coil structure. Indeed, mutations disrupting this putative structure abolished the dimerization capability and reduced the TAF-I activity in the Ad core DNA replication assay. Furthermore, we found that TAF-I mutants lacking the acidic tail act in a dominant-negative manner in this assay. These observations strongly suggest that the dimerization of TAF-I is important for its activity.

Template activating factor-I remodels the chromatin structure and stimulates transcription from the chromatin template.

To study the mechanisms of replication and transcription on chromatin, we have been using the adenovirus DNA complexed with viral basic core proteins, called Ad core. We have identified template activating factor (TAF)-I from uninfected HeLa cells as the factor that stimulates replication and transcription from the Ad core. The nuclease sensitivity assays have revealed that TAF-I remodels the Ad core, thereby making transcription and replication apparatus accessible to the template DNA. To examine whether TAF-I remodels the chromatin consisting of histones, the chromatin structure was reconstituted on the DNA fragment with core histones by the salt dialysis method. The transcription from the reconstituted chromatin was completely repressed, while TAF-I remodeled the chromatin and stimulated the transcription. TAF-I was found to interact with histones. Furthermore, it was shown that TAF-I is capable not only of disrupting the chromatin structure but also of preventing the formation of DNA-histone aggregation and transferring histones to naked DNA. The possible function of TAF-I in conjunction with a histone chaperone activity is discussed.

Cellular localization and expression of template-activating factor I in different cell types.

Template-activating factors I (TAF-I) alpha and beta have been identified as chromatin remodeling factors from human HeLa cells. TAF-I beta corresponds to the protein encoded by the set gene, which was found in an acute undifferentiated leukemia as a fusion version with the can gene via chromosomal translocation. To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I. The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells. The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain. It is of interest that the amounts of TAF-I alpha and beta vary among hemopoietic cells and some specific cell types do not contain TAF-I alpha. The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state. TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization. The localization of TAF-I in conjunction with the regulation of its activity is discussed.

NAP-I is a functional homologue of TAF-I that is required for replication and transcription of the adenovirus genome in a chromatin-like structure.

BACKGROUND: For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor-I (TAF-I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF-I also stimulates transcription from the Ad core DNA. RESULTS: Using mutant TAF-I proteins, we have demonstrated that the acidic stretch present in the carboxyl terminal region is essential for the stimulation of transcription from the Ad core. A genomic footprinting experiment with restriction endonuclease has revealed that TAF-I causes a structural change in the Ad core. TAF-I has been shown to have significant amino acid similarity to nucleosome assembly protein-I (NAP-I), which is involved in the formation of the chromatin structure. We have shown that TAF-I can be substituted by NAP-I in the activation of the cell-free Ad core transcription system. Two of the tripartite acidic regions and the region homologous to TAF-I in NAP-I are required for the maximal TAF-I activity of NAP-I. Furthermore, TAF-I has been shown to have NAP-I activity, and the acidic region of TAF-I is required for this activity. CONCLUSIONS: Since TAF-I causes the structural change of the Ad core and thereby activates transcription, TAF-I is thought to be one of the proteins which is involved in chromatin remodeling. NAP-I is structurally related to TAF-I and functionally substitutes for TAF-I. Furthermore, TAF-I has NAP-I activity. These observations suggest that this type of molecule has dual functions, possibly by participating in facilitating the assembly of the chromatin structure as well as perturbing the chromatin structure to allow transcription to proceed.

Stimulation of DNA transcription by the replication factor from the adenovirus genome in a chromatin-like structure.

Adenovirus (Ad) genome DNA is complexed with viral core proteins in the virus particle and in host cells during the early stages of infection. This DNA protein complex, called Ad core, is thought to be the template for transcription and DNA replication in infected cells. The Ad core functioned as template for DNA replication in the cell-free system consisting of viral replication proteins, uninfected HeLa nuclear extracts, and a novel factor, template activating factor-I (TAF-I) that we have isolated from uninfected HeLa cytoplasmic fractions. The Ad core did not function as an efficient template in the cell-free transcription system with nuclear extracts of uninfected HeLa cells. The addition of TAF-I resulted in the stimulation of transcription from E1A and ML promoters on the Ad core. TAF-I was required, at least, for the formation of preinitiation complexes. These observations suggest that, in addition to factors essential for transcription on naked DNA template, the factor such as TAF-I needed for replication of the Ad core is also required for transcription from the Ad genome in a chromatin-like structure.