Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3 -noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.The human parainfluenza virus type 3 (HPIV3), 1 belonging to the paramyxovirus family, is one of the major causes of pneumonia and bronchiolitis in infants (1). HPIV3 contains a negative strand RNA genome that is encapsidated by a nucleocapsid protein NP (68 kDa) and tightly associated with two RNA polymerase subunits, a large protein L (251 kDa) and a phosphoprotein P (90 kDa), to form the viral ribonucleoprotein (RNP) core (2, 3). The encapsidated genome RNA serves as a template for transcription to synthesize a leader RNA and six mRNAs as well as in replication to synthesize full-length genome RNA, both mediated by the viral RNA-dependent RNA polymerase. Recent studies demonstrate that participation of specific cellular proteins is critical for the regulation of gene expression of HPIV3 (4, 5). Protein kinase C-has been implicated in the phosphorylation of the virion-associated RNA polymerase subunit, the phosphoprotein P (5). Introduction of protein kinase C--specific peptide inhibitor in cultured cells abrogated HPIV3 replication providing strong evidence that protein kinase C-is involved in the HPIV3 life cycle (5). Another cellular protein, actin, was found to be required in transcription of purified viral RNP in vitro and was found to be involved in maintaining a moderately coiled structure of the RNP that appeared to facilitate transcription of the genome RNA by the RNA polymerase (4). The productive infection of HPIV3, thus, appears to require a close encounter between the viral genome and several cellular proteins. A detailed search of such putative cellular proteins and their characterization would lead to better understanding of their roles in the regulation of the intricate steps in viral gene expression.Sequence analysis of HPIV3 genome RNA reveals the presence of a sequence element at the 3Ј-end that serves as the binding site o...

Human parainfluenza viruses (HPIVs) are members of the paramyxovirus family (1, 2). Understanding the molecular events that control HPIV replication is particularly important because they are significant human pathogens causing diseases such as croup, pneumonia, and bronchiolotis in children (3). No suitable vaccines are currently available for these classes of viruses. HPIV type 3 (HPIV3), most pathogenic among the HPIVs, contains a linear genomic RNA (15,461 nt) of negative polarity and three structural proteins, NP (68-kDa nucleocapsid protein), L (251-kDa large protein), and P (90-kDa phosphoprotein), all ofwhich are enclosed within a lipid-containing envelope (1, 2). The L and P proteins together constitute the viral RNA-dependent RNA polymerase complex that, similar to that of other paramyxoviruses, transcribes the NP-bound genomic RNA (4-6); L is the RNA polymerase, whereas P is a required transcription factor or transactivator of L. Host cytoskeletal proteins can act as positive regulators in viral mRNA synthesis-e.g., actin in HPIV3 (7) or tubulin in Sendai and measles viruses (8, 9).Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is believed to be mediated by cellular protein kinases and is an important step in the formation of the active RNA polymerase complex (10-12). Cellular casein kinase II (CKII) has been implicated in phosphorylation of P proteins of vesicular stomatitis virus (VSV) (10) and respiratory syncytial virus (11,12). In the well-studied VSV system, use of unphosphorylated P protein (Po) obtained from a cDNA-derived expression in Escherichia coli has demonstrated that a cascade phosphorylation pathway is operative for Pprotein activation in vitro (13). The first step in the pathway is the phosphorylation mediated by cellular CKII, leading to aThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. structural alteration of the P protein (P1 form). The second step involves further phosphorylation of P1 by the protein kinase activity associated with the L protein, leading to the formation of the fully phosphorylated P2 form. This phosphorylation pathway is obligatory for the activation of P protein to function in the RNA polymerase complex. Here we report that HPIV3, in contrast, selects a specific isoform (C) of cellular protein kinase C (PKC-C) for phosphorylation of its P protein and also packages the same isoform within the virion.

Several studies indicate that paramyxoviruses require a specific cellular factor(s) for transcription of their genomic RNAs. We previously reported that the cellular cytoskeletal protein actin, in its polymeric form, participates in the transcription of human parainfluenza virus type 3 (HPIV3) in vitro. In the present study, we investigated the role of the polymeric form of actin, i.e., the actin microfilaments of the cytoskeletal framework, in the reproduction of HPIV3 in vivo. Pulse-chase labeling analyses indicate that the viral nucleocapsid-associated proteins, NP and P, are present predominantly in the cytoskeletal framework during infection. By in situ hybridization, we found that viral mRNAs and genomic RNA were synthesized from the nucleocapsids that were bound to the cytoskeletal framework. Double immunofluorescent labeling and confocal microscopy of the cytoarchitecture revealed that the viral nucleocapsids are specifically localized on the actin microfilaments. Treatment of cells with the actin-depolymerizing agent, cytochalasin D, resulted in the inhibition of viral RNA synthesis and ribonucleoprotein accumulation. These results strongly suggest that actin microfilaments play an important role in the replication of HPIV3.

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Cells of the innate immune system regulate immune responses through the production of antimicrobial peptides, chemokines, and cytokines, including human beta-defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human oral epithelial cell (HOEC) production of CCL20 and hBDs in response to Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 6 h, following stimulation by F. nucleatum cell wall (FnCW). The kinetics of CCL20 release is distinct from that of hBD-2 and -3, which peaks after 24 h and 48 h of FnCW stimulation, respectively. FnCW-induced release of CCL20 by HOECs requires both transcriptional and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, suggesting that it is secreted through a vesicle transport pathway. Other epithelium-derived agents that FnCW induces, such as hBD-2, hBD-3, tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤), are also able to release CCL20. By focusing on mitogen-activated protein kinases, we show that both extracellular signalregulated kinase 1/2 and p38, but not JNK, are required for hBD-, TNF-␣-, and IL-1␤-induced secretion of CCL20 by HOECs. The ability of FnCW and its induced hBDs to produce proinflammatory cytokines and CCL20 suggests the broad role of F. nucleatum and human antimicrobial peptides in primary immune responses elicited by oral epithelium.