Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan1
Author for correspondence: Yukihiro Nishiyama. Fax +81 52 744 2452. e-mail ynishiya{at}med.nagoya-u.ac.jp
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Abstract |
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This study focused on the effects of herpes simplex virus (HSV) infection on mitochondria. Since mitochondria play prominent roles in supplying energy, regulating calcium levels and controlling apoptotic cell death, processes that are critically important for cell mortality, it is important to study virus-mediated mitochondrial changes. Recently, it has been reported that mitochondria migrate to the virus assembly site in African swine fever virus (ASFV)-infected cells, where the respiratory function of mitochondria is activated (Rojo et al., 1998 ). This study suggested that mitochondria are altered to supply energy effectively for the virus morphogenetic process. It has also been shown that the herpesvirus saimiri-encoded Bcl-2 homologue prevents apoptosis by maintaining mitochondrial membrane potential and inhibiting the release of cytochrome c into the cytosol (Derfuss et al., 1998
). As for HSV, it was previously reported that synthesis of mitochondrial proteins in infected cells decreases progressively, dropping to about 60% (Latchman, 1988
; Lund & Ziola, 1986
). Tsurumi & Lehman (1990)
have reported that mitochondrial RNA polymerase appears in extracts of HSV-infected cells as a result of infection-induced disruption of the mitochondrial membrane, followed by leakage of the enzyme into the cytoplasm. In addition, Lund & Ziola (1985)
have observed that mitochondria in cells infected with HSV-1 exhibit a gradual decline in the initial Ca2+ uptake rate, dropping to 65% of the control rate at the end of the 12 h lytic cycle.
We first examined changes in mitochondrial subcellular localization by using immuno-fluorescent microscopy. In mock-infected Vero cells, rod-like mitochondria were distributed throughout the cells (Fig. 1a). When Vero cells were infected with HSV-2 186, they appeared to gather in the perinuclear region of the cytoplasm at 6 h post-infection (p.i.) (Fig. 1d
), and sometimes formed a ring-like structure (Fig. 1g
). The shape of mitochondrial particles in HSV-infected cells became more unclear. Similar observations were made in HSV-1 (KOS)-infected Vero and HEp-2 cells (not shown).
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The appearance of assembled mitochondria reminded us of the stress-responsive changes caused by heat shock (Collier et al., 1993 ). It has been shown that some stress-responsive cellular genes are induced by HSV infection (Notarianni & Preston, 1982
; La Thangue & Latchman, 1988
). We therefore performed Western blot analysis of the chaperonin HSP60, which resides exclusively in mitochondria and plays a major role in the folding and assembly of proteins conveyed from the cytoplasm into the mitochondria (Martin et al., 1995
). HEp-2 cells were mock infected or infected with HSV-2 186 at an m.o.i. of 5. Western blotting was carried out as described previously (Daikoku et al., 1993
). As expected, infection with HSV-2 resulted in significant induction of HSP60 (Fig. 2a
).
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In order to estimate mitochondrial energy supply function, the content of ATP was determined by the ATP assay system (Toyo ink. Co.), in which luciferinluciferase reactions were used, since these reactions reflect ATP content. From 3 to 9 h p.i., HEp-2 cells infected with HSV maintained the level of ATP (100 and 102, 111 and 103 and 96 and 106% of mock infection levels, in 186 and KOS cells at 3, 6 and 9 h p.i., respectively), but ATP declined significantly from 12 h p.i. (53 and 67, 51 and 44, 45 and 42% at 12, 15 and 18 h p.i.) (Fig. 2c).
Cells can also generate ATP by glycolysis, starting with glucose and converting it into lactate. Although the reactions are carried out in the cytoplasm and not in mitochondria, production of lactate would be an indirect measure of the capacity of mitochondria to generate ATP. Analysis of the lactate secreted into the cell culture medium showed no significant increase or decrease at 6 h p.i. (Fig. 2d), suggesting that mitochondria maintained their energy supply function until the middle stage of infection.
Proton translocation by the electron transport system results in the highly negative potential of the inner mitochondrial membrane. Since rhodamine 123 is an aromatic cation, it distributes electrophoretically into the mitochondrial matrix across the inner membrane in response to membrane potential. Agents known to depolarize or deenergize mitochondria, such as uncouplers or respiratory inhibitors, decrease rhodamine 123 fluorescence of mitochondria in cells (Johnson et al., 1981 ). Fluorescence is also reduced when cells are involved in apoptotic cell death processes (Shimizu et al., 1996
). Hence, we used rhodamine 123 to monitor mitochondrial membrane potential. The results of flow cytometric analysis of rhodamine 123 in HSV-infected HEp-2 cells are shown in Fig. 2(e)
(h)
. At 6 h p.i., membrane potential was not altered or was slightly reduced by HSV infection (Fig. 2f
). However, at 12 and 18 h p.i., HSV infection resulted in a significant decrease in membrane potential (Fig. 2g
, h
). The small peak of HSV-2-infected cells at 18 h p.i. was thought to represent dead cells, since a certain population of HEp-2 cells infected with HSV-2 are known to be apoptotic (Koyama et al., 1998
). As a control, actinomycin D-treated cells, in which apoptosis was markedly induced, were prepared so that we were able to learn the relative level of the reduction in HSV-infected cells (Fig. 2e
). We therefore conclude that, while HSV infection caused a significant reduction in membrane potential in the late stage of infection, the potential was maintained until at least 6 h p.i.
There is a consensus that mitochondria are scattered or transported in association with cytoskeletal elements such as microtubules or intermediate filaments. Microtubules have been shown to play a central role in the transportation of mitochondria (Heggeness et al., 1978 ; Miller & Lasek, 1985
), although intermediate filaments are also reported to be involved in mitochondrial migration (Collier et al., 1993
). In order to clarify which structures were crucial for the accumulation of mitochondria and tegument proteins in HSV-infected cells, we used the following inhibitors: nocodazole and vinblastine to disassemble the tubulin network and taxol to stabilize microtubules. At 9 h p.i., cells were fixed with acetone and then mitochondria and UL41 were stained by rhodamine and FITC, respectively. In mock-infected cells (Fig. 3a
), no green fluorescence was detectable but a number of small red dots representing mitochondria were observed in the cytoplasm. The addition of DMSO, which was used to dissolve the drugs, did not affect migration of the UL41 protein or mitochondria (Fig. 3b
). Nocodazole (Fig. 3c
) and vinblastine (Fig. 3d
) inhibited the migration of the UL41 product and mitochondria. In the presence of these drugs, the UL41 gene product was distributed in the cytoplasm as small dots and there was no apparent interaction between the distributions of mitochondria and UL41 protein. The subcellular localization of UL41 protein in HSV-2-infected cells that were treated with nocodazole and vinblastine (Fig. 3c
, d
) was similar to that in cells in which UL41 was singly expressed (Fig. 3f
). According to the PSORT program (Nakai & Horton, 1999
), the UL41 gene product is predicted to reside in microbodies (peroxisomes) or lysosomes in animal cells. It seems that the UL41 protein localized in microbodies or lysosomes as small dots in UL41-transfected cells, which suggests that other viral proteins may be required to facilitate accumulation of the UL41 protein to the region along the microtubule network. In contrast to the effects of nocodazole and vinblastine, treatment with taxol, which stabilizes microtubules, resulted in the migration of the UL41 protein and mitochondria to the region around the nucleus, although some mitochondria were left in the cytoplasm (Fig. 3e
). Control experiments showed that the microtubule network was disassembled effectively by nocodazole (Fig. 3h
) and was stabilized and thickened by taxol (Fig. 3i
), while treatment with the solvent, DMSO, had no effect on the microtubule network (Fig. 3g
).
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Microtubules are one of the cytoskeletal structures, consisting of tubulin and expanding from the centre to the periphery of the cell. Incoming HSV is transported to the nucleus along microtubules (Sodeik et al., 1997 ). Our results suggest that both the UL41 gene product and mitochondria were transported to the region around the nucleus along microtubules in HSV-infected cells. Some reports have pointed out the involvement of intermediate filaments in the migration of mitochondria, but we observed an inhibition of transport of mitochondria and UL41 protein with the addition of microtubule-disassembling drugs (Fig. 3c
, d
). Hence, we conclude that microtubules play an essential role in gathering mitochondria and UL41 protein.
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Acknowledgments |
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References |
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Received 7 July 1999;
accepted 8 October 1999.