Akihiro Hiraki

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.

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°.

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 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.

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.

Changes in fused cells induced by hvj (sendai virus): relationship of morphological changes to endocytosis of the surface membrane.

Fused Ehlrich ascites tumor (EAT) cells induced by hemagglutinating virus of Japan (HVJ; Sendai virus) had an irregular shape, reflecting the shape of cell aggregates before fusion. During subsequent culture, the fused cells gradually took on a spherical form within 60min. Examination of the fused cells revealed a vigorous endocytosis of the cell membrane during the morphological change. When EAT cells were treated with porphyrin derivatives, and the morphological change to a spherical form was inhibited, endocytosis of fused cells was also suppressed, suggesting that the change is closely associated with endocytotic activity. Further examination with porphyrin derivatives and hydrogen peroxide suggested that the inhibition of morphological change is due to the suppression of endocytosis by active oxygen species produced by these substances. Experiments using an endocytotic inhibitor, methylamine, indicated that endocytosis is essential for the morphological change that occurs in the fused cells.