Specific Phosphorylated Forms of Glyceraldehyde 3-Phosphate Dehydrogenase Associate with Human Parainfluenza Virus Type 3 and Inhibit Viral Transcription In Vitro

Abstract

). To gain further insight into these molecular interactions, we analyzed the virion-associated GAPDH and La protein using two-dimensional gel electrophoresis and immunoblotting. The GAPDH was resolved into two major and one minor molecular species migrating in the pI range of 7.6 to 8.3, while the La protein was resolved into five molecular species in the pI range of 6.8 to 7.5. The GAPDH isoforms present in the virions were also detected in the cytoplasmic fraction of CV-1 cell extract, albeit as minor species. On the other hand, the multiple molecular forms of La protein as seen within the virions were readily detected in the total CV-1 cell extract. Further analysis of virion-associated GAPDH by in vivo labeling with [ 32 P]orthophosphate revealed the presence of multiple phosphorylated species. The phosphorylated species were able to bind specifically to the viral cis-acting 3 genome sense RNA but failed to bind to the leader sense RNA, as determined by gel mobility shift assay. In contrast, the La protein isoforms present within the virions were not phosphorylated and bound to the viral cis-acting RNAs in a phosphorylation-independent manner. The GAPDH isoforms purified from the CV-1 cell cytoplasmic fraction inhibited viral transcription in vitro. Consistent with this, flag-tagged recombinant GAPDH synthesized by using the vaccinia virus expression system also inhibited viral transcription. Together, these data indicate that specific phosphorylated forms of GAPDH associate with HPIV3 and are involved in the regulation of virus gene expression.Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is second only to respiratory syncytial virus in causing severe respiratory tract infections in children and young adults (6). The single-stranded RNA genome of HPIV3, 15,461 nucleotides (nt) long, is contained within a helical nucleocapsid (15). Three virus-encoded proteins, the nucleocapsid protein N (68 kDa), the phosphoprotein P (90 kDa), and the RNA polymerase L (257 kDa), are associated with the nucleocapsid to form a transcribing ribonucleoprotein (RNP) complex (1, 15). The N protein enwraps the genome RNA, while the L and P proteins together constitute the RNA-dependent RNA polymerase complex that transcribes and replicates the N-bound genome RNA. During transcription, the RNA polymerase synthesizes a short leader RNA, followed by six capped and polyadenylated mRNAs. During replication, on the other hand, a full-length plus-strand copy of the genome RNA is synthesized which, in turn, serves as the template for synthesis of the minus-strand genome RNA (1, 6, 15). Studies from our laboratory, as well as others, indicate that cellular cytoskeletal proteins such as actin and tubulin are involved in the activation of transcription of several paramyxoviruses (4,9,12,22,34,35). Similarly, cellular RNA binding proteins that interact with the viral cis-acting elements are believed to be involved in the regulation of transcription and replication (25,26,28).The minus-sense genomic 3Ј end and its complementary l...

Moonlighting cell surface GAPDH recruits Apo Transferrin to effect iron egress from mammalian cells

“…Our LC-MS/MS results demonstrate that GAPDH recruited upon iron loading lacks phosphorylation and acetylation and also has a lower abundance of several PTMs, including oxidation, dimethylation and nitrosylation in comparison to GAPDH recruited upon iron depletion. These modifications are well known for their ability to shift the isoelectric point of proteins as observed by us in this study, as well as by numerous other workers previously (Choudhary et al, 2000;Grillon et al, 2012;Kuyumcu-Martinez et al, 2007;Madian et al, 2012;Park et al, 1988;Zhang et al, 2011). The presence of numerous PTMs in GAPDH has been well established in earlier reports (Seidler, 2013;Seo et al, 2008).…”

Parainfluenza Viruses

“…Similarly, human peripheral blood leukocytes stimulated by Sendai virus have been shown to produce macrophage inflammatory protein 1 alpha, monocyte chemotactic protein 1, tumor necrosis factor alpha, IL-6, and IL-8 (163). Interferon may inhibit HPIV-3 primary transcription (52), and likewise HPIV-3 may inhibit interferon-mediated pathways in T cells, including major histocompatibility complex class II antigen expression (108). Another inflammatory mediator induced by Sendai virus is intercellular cell adhesion molecule 1, which has been shown to be responsible for neutrophil and lymphocyte migration into the lung (334).…”

Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter

“…The contribution of leader RNA transcript to viral RNA synthesis is worthy of consideration. Interactions of leader RNA transcript with host proteins such as La protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and heterogeneous nuclear ribonucleoprotein were reported for vesicular stomatitis virus, rabies virus and hPIV3 (Kurilla et al, 1983(Kurilla et al, , 1984De et al, 1996;Gupta et al, 1998), and an involvement of GAPDH in hPIV3 transcription was suggested (Choudhary et al, 2000). We hypothesized that the intrinsic properties of leader RNA transcript from chimeric mini-genomes (and viruses) may affect viral gene expression.…”