Jeman Kim

Heat Shock Protein 70 Is Related to Thermal Inhibition of Nuclear Export of the Influenza Virus Ribonucleoprotein Complex

ABSTRACT The influenza virus genome replicates and forms a viral ribonucleoprotein complex (vRNP) with nucleoprotein (NP) and RNA polymerases in the nuclei of host cells. vRNP is then exported into the cytoplasm for viral morphogenesis at the cell membrane. Matrix protein 1 (M1) and nonstructural protein 2/nuclear export protein (NS2/NEP) work in the nuclear export of vRNP by associating with it. It was previously reported that influenza virus production was inhibited in Madin-Darby canine kidney (MDCK) cells cultured at 41°C because nuclear export of vRNP was blocked by the dissociation of M1 from vRNP (A. Sakaguchi, E. Hirayama, A. Hiraki, Y. Ishida, and J. Kim, Virology 306:244-253, 2003). Previous data also suggested that a certain protein(s) synthesized only at 41°C inhibited the association of M1 with vRNP. The potential of heat shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37°C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1. Moreover, NS2 accumulated in the nucleoli of host cells cultured at 41°C, showing that the export of NS2 was also disturbed at 41°C. However, NS2 was exported normally from the nucleus, irrespective of PGA1 treatment at 37°C, suggesting that HSP70 does not influence NS2.

Nuclear export of influenza viral ribonucleoprotein is temperature-dependently inhibited by dissociation of viral matrix protein

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells.

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ‐mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ‐induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.

Temperature-Sensitive Viral Infection: Inhibition of Hemagglutinating Virus of Japan (Sendai Virus) Infection at 41°

While investigating myoblast fusion using enveloped viruses, we unexpectedly found that the production of hemagglutinating virus of Japan (HVJ; Sendai virus) was suppressed temperature dependently in quail myoblasts transformed with a temperature-sensitive Rous sarcoma virus, which proliferate at 35.5° but differentiate at 41°; viral production was normal at 35.5° but suppressed at 41° irrespective of the species of host cells. The production of some viruses, i.e. measles virus, influenza virus, herpes simplex virus type 1 and poliovirus, was also markedly suppressed at 41°, suggesting that a temperature of 41° affects viral infection generally. To clarify the mechanism of the suppression, the infectious pattern of HVJ was examined both at 37° and at 41° in LLC-MK2 cells. The synthesis of HVJ-specific proteins was inhibited at the transcriptional level at 41°, although viral penetration by envelope fusion was not affected. The transcriptional inhibition was also seen in quail fibroblasts, which do not express a 70-kD heat shock protein (HSP70), suggesting that HSP70 is dispensable for the inhibition of viral gene transcription at 41°. Further, when the infected cells were incubated at 41° after the viral proteins had been synthesized at 37°, viral production was also inhibited. Immunofluorescent staining of the cells exposed to 41° showed that HVJ envelope proteins formed large aggregates on the cell surface, into which both M and NP proteins were assembled. Under the electron microscope, HVJ virions appeared normal even at 41°, but were detected in clusters on the cell surface, unlike at 37°. These observations suggested that the release of HVJ virions from the cell surface was inhibited for some reason at 41°. Consequently, it was indicated that two steps, viral gene transcription and the release of virions, were inhibited at 41°.

Dynamic distribution of an antigen involved in the differentiation of avian myoblasts: II. possible association of ?1 integrin with myofibril organization

Previous studies have shown that a monoclonal antibody, H-145, inhibits myotube formation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) [Hyodo and Kim, 1994: Exp. Cell Res. 212:120-131]. The antigen recognized by H-145 (H-145 antigen), which is a glycoprotein with a molecular mass of about 116 kDa, is related to a step immediately before myoblast fusion. To determine the functional significance of H-145 antigen, we examined its dynamic state during myogenic differentiation of QM-RSV cells. H-145 antigen showed a unique and discrete distribution. In immature myotubes immediately after myoblast fusion, many ring-like structures of H-145 antigen appeared on the ventral surface of the cells, encircling the actin dots detected simultaneously by immunofluorescence and interference reflection microscopy. The core of the ring-like structures was filled with the termini of actin bundles, mainly formed by alpha-actin. Other cytoskeletal-associated proteins, such as vinculin and alpha-actinin, were also associated with these structures. The ring-like structures of H-145 antigen were observed only during a restricted period when myoblasts fused actively, suggesting their relationships to myotube formation and an early stage of myofibril formation. With maturation of the myotubes, most of the H-145 antigen became redistributed in linear arrays on the apical cell surface and was probably associated with the termini of actin bundles to organize myofibrils, suggesting that the antigen was also related to maturation of myotubes. Experiments using monoclonal antibodies against chick beta1 integrin showed that H-145 antigen is beta1 integrin or a very closely related derivation. Thus H-145 antigen (beta1 integrin) is possibly involved in both myoblast fusion and the myofibril organization in myotubes.

Antiviral substance from silkworm faeces : Characterization of its antiviral activity

The antiviral activity of a substance (L4–1) purified from silkworm faeces was examined in an HVJ (Sendai virus)—LLC‐MK2 cell system. Its antiviral effect depended on the period of light irradiation and was inhibited by sodium sulfite and anaerobic conditions. These results indicate that the antiviral activity of L4–1 is associated with active oxygen species produced from the substance. SDS‐polyacrylamide gel electrophoretic analysis showed that viral proteins were damaged by this substance under light irradiation. The results suggest that the antiviral activity is due to damage to viral protein(s) caused by active oxygen species produced from L4–1.

Characterization of heterokaryons between skeletal myoblasts and preadipocytes: myogenic potential of 3T3-L1 preadipocytes

It has been shown previously that heterokaryons between myoblasts and non-myogenic cells disturb myogenic differentiation (Hirayama et al. (2001); Cell Struct. Funct. 26, 37-47), suggesting that some myogenesis inhibitory factors exist in non-myogenic cells. Skeletal myoblasts and adipose cells are derived from a common mesodermal stem cell, indicating that both cells have a closer relationship in the developmental lineage than the other somatic cells. To investigate the functional relationship between myoblasts and adipose cells, heterokaryons between quail myoblasts and 3T3-L1 cells, a mouse preadipocyte cell line, were prepared and examined for characteristics of myogenic differentiation. Myogenic differentiation was inhibited in the heterokaryons between quail myoblasts and well-differentiated (adipocytes) 3T3-L1 cells. On the contrary, normal myogenic differentiation proceeded in the heterokaryons between quail myoblasts and undifferentiated (preadipocytes) 3T3-L1 cells. Further investigation showed that the mouse myogenin gene from 3T3-L1 cells was transactivated in the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells. The results demonstrated that undifferentiated 3T3-L1 cells have no myogenesis inhibitory factors but acquire these during terminal differentiation into adipocytes.

Characterization of Temperature-Sensitive HVJ (Sendai Virus) Infection in Vero Cells: Inhibitory Mechanism of Viral Production at 41°

In a previous study, it was found that the synthesis of hemagglutinating virus of Japan (HVJ; Sendai virus)-specific proteins was inhibited at the transcriptional level at 41° in LLC-MK2 cells. During an investigation of the temperature sensitivity of HVJ production in other host cells, the synthesis of HVJ-specific proteins was recognized even at 41° in Vero cells. Viral production, however, was not detected, indicating the inhibition of steps after the synthesis of viral proteins. Hemadsorption activity was not detected at 41°, suggesting problems with the envelope proteins, especially hemagglutinin-neuraminidase (HN) protein, at the cell membrane. Immunofluorescent staining and surface immunoprecipitation showed that HN protein was not present on the surface in spite of its localization in the cytoplasm. Further, analysis of the cell membrane fraction suggested that fusion (F) protein was integrated into the cell membrane but HN protein was not at 41°. Electron microscopic observation showed that budding sites with spike structures formed and nucleocapsids assembled under the sites at 41° without HN protein, although budded HVJ virions were not detected. At this time, F protein was exposed to the cell membrane and interacted with matrix and nucleocapsid proteins. The results suggested that the suppression of HVJ production at 41° was due to the absence of HN protein in the membrane of Vero cells.

CHARACTERIZATION OF MYOGENIC CELL MEMBRANE: SPONTANEOUS FORMATION OF HETEROKARYOTIC MYOTUBES BETWEEN TWO DIFFERENT KINDS OF MYOBLASTS

In a previous study, it has been shown that presumptive mouse C2 myoblast cells are strongly resistant to HVJ (hemaglutinating virus of Japan, Sendai virus)‐mediated cell fusion, but do become capable of fusion upon differentiation. Quail myoblasts transformed with a temperature‐sensitive mutant of Rous sarcoma virus (QM‐RSV cells) also become more sensitive to HVJ‐mediated cell fusion during differentiation. Investigations were undertaken to see whether heterokaryotic myotubes were formed spontaneously by co‐culture of two different kinds of myogenic cells, QM‐RSV cells and C2 cells. When both cells were committed to myotube formation, they spontaneously fused without HVJ on co‐culture. On the other hand, when both or one of the cells were in the presumptive state, heterokaryons were not formed by co‐culturing. Furthermore, committed QM‐RSV cells did not fuse with non‐myogenic cells. These results indicate that the membranes of myogenic cells change to become capable of fusion for myotube formation during differentiation.

Changes in fused cells induced by hvj (sendai virus): redistribution of cytoplasmic organelles and cytoskeletal reorganization.

To understand the relationship between the location of organelles and cellular function, we examined the dynamic state of cytoplasmic organelles and cytoskeleton in polynuclear Ehrlich ascites tumor (EAT) cells fused with hemagglutinating virus of Japan (HVJ; Sendai virus) by confocal laser scanning microscopy. Irregular fused cells gradually became spherical during culture, and nuclei and mitochondria were redistributed in the fused cell; nuclei formed a cluster surrounded by mitochondria. F‐actin, vimentin, and microtubules were also reorganized with the redistribution of cell organelles. Further, when the morphological change was inhibited by L4‐1, a chlorophyll‐like substance derived from silkworm faeces, or pyropheophorbide‐a, the arrangement of organelles and cytoskeleton remained disturbed, suggesting that the movement of the cytoskeleton is closely associated with cell shape and the distribution of cytoplasmic organelles.