A. Hajime Koyama

Physiological significance of apoptosis in animal virus infection

In contrast to insect viruses, animal viruses can produce considerable amounts of progeny virus in cells undergoing apoptosis. Nevertheless, viruses in general have acquired the ability to escape apoptosis of infected cells. These facts indicate that the role of apoptosis in virus infection is different in insect virus and animal virus, although both viruses need to avoid apoptosis of the infected cells for a viral life cycle in nature. In animal virus infection, the primary role of apoptosis is considered not to be a premature lysis of the infected cells (and the following abortion of virus multiplication) but to allow the dying cells to be phagocytosed by macrophages. This phagocytosis is able to prevent dysregulated inflammatory reactions at the site of virus infection and to initiate a specific immune response against the infected virus.

Induction of apoptotic DNA fragmentation by the infection of vesicular stomatitis virus

Vesicular stomatitis virus (VSV) induced apoptosis of infected HeLa cells. Fragmentation of chromosomal DNA into nucleosomal oligomers as well as a characteristic nuclear fragmentation were observed in the infected cells. The kinetics of the apoptotic response was determined in relation to viral multiplication and cytopathic effect. Comparison of these kinetics revealed that the production of progeny virus occurs at almost similar kinetics to that of the DNA fragmentation, so that viral multiplication is not interrupted by the apoptotic cell damage.

Antiapoptotic activity of herpes simplex virus type 2 : the role of US3 protein kinase gene

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.

The HIV-1 Vpr displays strong anti-apoptotic activity

Mutations in the human immunodeficiency virus type 1 (HIV‐1) vpr gene only slightly reduce the replication rate of the virus. To study the role of HIV‐1 Vpr in biological effects on cells, HEp‐2 cells, which express HIV‐1 Vpr constitutively but at a low level, were established. While control HEp‐2 cells underwent apoptosis when incubated with sorbitol, the morphological and biochemical apoptotic changes were inefficiently induced in the HIV‐1 Vpr‐expressing cells by the same treatment. These results clearly indicate that HIV‐1 Vpr has anti‐apoptotic activity, and raise the possibility that Vpr acts as a weak activator of virus replication through anti‐apoptosis.

Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells

Mutants of human immunodeficiency virus type 2 (HIV-2) carrying a frame-shift mutation in vpx, vpr, and in both genes were monitored for their growth potentials in a newly established lymphocytic cell line, HSC-F. Worthy of note, the replication of a vpx single mutant, but not vpr, was severely impaired in these cells, and that of a vpx-vpr double mutant was more damaged. Defective replication sites of the vpx single and vpx-vpr double mutants were demonstrated to be mapped, respectively, to the nuclear import of viral genome, and to both, this process and the virus assembly/release stage. While the mutational effect of vpr was small, the replication efficiency in one cycle of the vpx mutant relative to that of wild-type virus was estimated to be 10%. The growth phenotypes of the vpx, vpr, and vpx-vpr mutant viruses in HSC-F cells were essentially repeated in primary human lymphocytes. In primary human macrophages, whereas the vpx and vpx-vpr mutants did not grow at all, the vpr mutant grew equally as well as the wild-type virus. These results strongly suggested that Vpx is critical for up-regulation of HIV-2 replication in natural target cells by enhancing the genome nuclear import, and that Vpr promotes HIV-2 replication somewhat, at least in lymphocytic cells, at a very late replication phase.

The Mode of Entry of Herpes Simplex Virus Type 1 into Vero Cells

The mode of entry of herpes simplex virus type 1 (HSV‐1) into Vero cells was investigated quantitatively with biological techniques. The entry of virus occurred rapidly when the virus‐adsorbed cells were incubated at 37 C. The kinetics of virus entry was found to be similar to that of the process of uncoating, indicating that the uncoating of HSV‐1 occurs simultaneously with the entry of virus into the cell. Experiments with ammonium chloride revealed that acidity in endosomes is not necessary for the entry or uncoating of HSV‐1, in contrast with the cases of enveloped RNA viruses. In addition, endocytosis of the virus seems to be one of the processes of entry for HSV‐1. However, the kinetics of endocytosis showed that the cell‐bound virus is endocytosed gradually and suggested that the endocytosis of HSV‐1 does not lead the virus to an uncoating process. These results are most consistent with a mechanism of entry for HSV‐1 involving fusion of the viral envelope with the plasma membrane of the host cell.

Arginine Facilitates Inactivation of Enveloped Viruses

Virus inactivation is a key step for the purification of pharmaceutical proteins derived from recombinant mammalian expression systems and conventionally done using low pH-treatment, which is often harmful to the proteins to be purified. This is particularly true for antibodies, because immunoglobulin proteins undergo conformational changes at acidic pH. We have been developing mild elution solvents using arginine for Protein-A chromatography to minimize the low pH-induced damages on the antibodies. Here we have tested the aqueous solutions containing arginine or butyroyl-arginine at or above pH 4.0 for their effects on virus inactivation, since these solvents are effective above pH 4.0 in elution of bound antibodies from Protein-A columns. When the virus was incubated on ice, 0.1 M sodium citrate was totally ineffective above pH 4.0, but aqueous solutions containing arginine above 0.35 M or butyroyl-arginine above 0.28 M showed extensive virus killing at or even above pH 4.0.

Solubility enhancement of gluten and organic compounds by arginine

Arginine suppresses protein-protein and protein-surface interactions and thus is expected to increase the solubility of the proteins. We have examined here the effects of arginine on the solubility of a highly insoluble protein, gluten, and two organic compounds, octyl-gallate and coumarin, which have low to moderate aqueous solubilities. Arginine significantly increased the solubility of these molecules concentration dependently, while a weak salting-out salt, NaCl, decreased it. The observed ability of arginine to salt-in these compounds can be explained from its binding to aromatic groups and protein surface. Such solubilizing action of arginine may be used to enhance the solubility of poorly soluble organic drug substances.

The effect of ammonium chloride on the multiplication of herpes simplex virus type 1 in Vero cells

The multiplication of herpes simplex virus type 1 (HSV-1) in Vero cells is inhibited by ammonium chloride. The formation of infectious virus was inhibited immediately after the addition of the agent into the culture fluid and was restored by removal of the agent. Although neither viral DNA replication nor nucleocapsid formation were affected by the addition of ammonium chloride at 4 h postinfection, the agent markedly inhibited the formation of enveloped particles and completely the formation of infectious progeny virus. These results indicate that one of the effects of ammonium chloride on the multiplication of HSV-1 is the inhibition of envelopment of viral nucleocapsids. In addition, the envelopment of HSV-1 nucleocapsids was inhibited immediately after the addition of monensin into the culture fluid. These findings suggest the importance of acidic pH of an intracellular compartment in the envelopment of HSV-1.

Inhibition of multiplication of herpes simplex virus by caffeic acid

Hot water extracts of coffee grinds and commercial instant coffee solutions have been shown to exhibit marked antiviral and virucidal activities against herpes simplex virus type 1 (HSV-1). Specifically, it has been shown that caffeine and N-methyl-pyridinium formate inhibit the multiplication of HSV-1 in HEp-2 cells. The present study examined the virological properties and the antiviral activity of caffeic acid against HSV-1. Caffeic acid inhibited the multiplication of HSV-1 in vitro, while chlorogenic acid, a caffeic acid ester with quinic acid, did not. These reagents did not have a direct virucidal effect. The one-step growth curve of HSV-1 showed that the addition of caffeic acid at 8 h post infection (h p.i.) did not significantly affect the formation of progeny viruses. An analysis of the influence of the time of caffeic acid addition, revealed that addition at an early time post infection remarkably inhibited the formation of progeny infectious virus in the infected cells, but its addition after 6 h p.i. (i.e., the time of the completion of viral genome replication) did not efficiently inhibit this process. These results indicate that caffeic acid inhibits HSV-1 multiplication mainly before the completion of viral DNA replication, but not thereafter. Although caffeic acid showed some cytotoxicity by prolonged incubation, the observed antiviral activity is likely not the secondary result of the cytotoxic effect of the reagent, because the inhibition of the virus multiplication was observed before appearance of the notable cytotoxicity.