Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

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Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90).In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication.IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a mature polymerase complex with the polymerase cofactor of L, phosphoprotein. Hsp70 collaborates with Hsp90 to regulate biogenesis of the MuV L protein. The functions of these chaperones on the viral polymerase may be common among paramyxoviruses because the L protein of measles virus is also similarly regulated. Our data provide important insights into the molecular mechanisms of paramyxovirus polymerase maturation as well as a basis for the development of novel antiviral drugs.KEYWORDS heat shock protein 90, large protein, mumps virus, polymerase M umps is a common childhood illness characterized by painful swelling of the parotid glands and is often accompanied by severe complications such as orchitis, aseptic meningitis, pancreatitis, and deafness (1). Mumps virus (MuV), which belongs to the genus Rubulavirus of the family Paramyxoviridae and the order Mononegavirales, is the causative agent of mumps (2). The viral genome consists of a

Section: Discussionsupporting

confidence: 48%

Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation

Katoh

Kubota

Nakatsu

et al. 2017

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“…Many mononegaviruses, including vesicular stomatitis virus (41), rabies virus (42), ebolavirus (43), and mumps virus (44), produce cytoplasmic inclusion bodies (equivalent to the MV RNP complex) where viral RNA synthesis occurs. In vesicular stomatitis virus-infected cells, primary RNA synthesis takes place throughout the host cell cytoplasm, and viral protein expression redirects viral RNA synthesis to cytoplasmic inclusions (41).…”

Section: Discussionmentioning

confidence: 99%

Actin-Modulating Protein Cofilin Is Involved in the Formation of Measles Virus Ribonucleoprotein Complex at the Perinuclear Region

Koga

Sugita

Noda

et al. 2015

In measles virus (MV)-infected cells, the ribonucleoprotein (RNP) complex, comprised of the viral genome and the nucleocapsid (N) protein, phosphoprotein (P protein), and large protein, assembles at the perinuclear region and synthesizes viral RNAs. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, interacts with the MV N protein and aids in the formation of the RNP complex. Knockdown of cofilin using the short hairpin RNA reduces the formation of the RNP complex after MV infection and that of the RNP complex-like structure after plasmid-mediated expression of MV N and P proteins. A lower level of formation of the RNP complex results in the reduction of viral RNA synthesis. Cofilin phosphorylation on the serine residue at position 3, an enzymatically inactive form, is increased after MV infection and the phosphorylated form of cofilin is preferentially included in the complex. These results indicate that cofilin plays an important role in MV replication by increasing formation of the RNP complex and viral RNA synthesis. IMPORTANCE Many Measles is a highly contagious viral disease characterized by high fever, respiratory symptoms, conjunctivitis, and a macropapular rash. The patients exhibit immunosuppression, often resulting in secondary infections, the main cause of measles-associated mortality and morbidity. On rare occasions, measles virus (MV) persists in the central nervous system and causes subacute sclerosing panencephalitis with long incubation periods (1). Although mortality and morbidity have been greatly reduced thanks to worldwide vaccination efforts, the World Health Organization estimates that 145,700 people died of measles in 2013 (http://www .who.int/mediacentre/factsheets/fs286/en/).MV, a member of the genus Morbillivirus in the family Paramyxoviridae, possesses 6 genes coding for the nucleocapsid (N) protein, phosphoprotein (P protein), matrix (M) protein, and hemagglutinin (HA), fusion, and large proteins. The two envelope glycoproteins, the hemagglutinin and fusion proteins, mediate receptor binding and membrane fusion, respectively (1). Pathogenic MVs use as cellular receptors the signaling lymphocyte activation molecule (SLAM) (2, 3) and nectin 4 (4, 5), which are expressed on immune and epithelial cells, respectively. The N, P, and large proteins, together with the viral genome, constitute the ribonucleoprotein (RNP) complex, which is found at the perinuclear region in MV-infected cells (6-8) and functions as the RNAdependent RNA polymerase. The M protein is involved in virion assembly and the budding process (1).Besides the P protein, the MV P gene encodes two additional proteins, C and V (9, 10). The V mRNA is produced through the insertion of a single nontemplated guanine at the editing site of the P gene during the transcription. The V protein possesses 231 amino acid residues in the N terminus which are identical to those possessed by the P protein and 68 unique resid...

“…Nocodazole was purchased from Sigma (St. Louis, MO). Anti-N (clone 23D), -M (clone 79D), and -HN (clone 78) mouse monoclonal antibodies (MAbs) and anti-MuV N rabbit polyclonal antibody (PAb) were prepared as described previously (33)(34)(35). Anti-␤actin (clone AC-15) and anti-␣-tubulin (clone DM1A) mouse MAbs were purchased from Sigma.…”

Section: Cells and Virusmentioning

confidence: 99%

Mumps Virus Is Released from the Apical Surface of Polarized Epithelial Cells, and the Release Is Facilitated by a Rab11-Mediated Transport System

Katoh

Nakatsu

Kubota

et al. 2015

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Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients.In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection. IMPORTANCE Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection.However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV. Mumps is a common childhood illness characterized by painful swelling of the parotid glands and is often accompanied by severe complications such as orchitis, aseptic meningitis, pancreatitis, and deafness (1). However, despite the prevalence and seriousness of the disease, the molecular basis of the pathogenesis of mumps is still poorly understood.Mumps virus (MuV), which belongs to the genus Rubulavirus within the family Paramyxoviridae, is the causative agent of mumps (2). The virus infection of cells is initiated by the binding of the hemagglutinin-neuraminidase (HN) protein to sialic acids of the cell surface (3). After receptor binding, the fusion (F) protein induces pH-independent fusion of the viral envelope with the host plasma membrane, and the viral genomic RNA is released into the cytoplasm (4). The viral genomic RNA encapsidated by the nucleocapsid (N) protein forms an active template for RNA replication and transcription, a viral ribonucleoprotein (vRNP), with viral polymerases composed of the phosphoprotein (P protein) and large (L) protein (5...