Hiizu Aoki

Mechanism of Uncoating of Influenza B Virus in MDCK Cells: Action of Chloroquine

Exposure of influenza B virus-infected MDCK cells to chloroquine at the time of infection resulted in significant inhibition of infection. The appearance of input virus in the intracellular vesicles was not affected in the presence of the drug, but primary transcription of the virus genome did not occur. Chloroquine caused a rapid rise in the pH inside the lysosomes of MDCK cells, to 6.5 from the physiological pH 5.6. In contrast, exposure of infected cells incubated in acidic medium (pH 6.0) to chloroquine did not cause an increase in lysosomal pH and this low pH treatment during the chloroquine-sensitive phase was followed by virus production. Influenza B virus induced haemolysis of chick erythrocytes at low pH values (5.0 to 5.9) which was associated with cell-cell membrane fusion. It is likely that chloroquine prevents the uncoating of influenza B virus by increasing the lysosomal pH above the critical value required for inducing fusion between the virus envelope and the lysosomal membrane.

Protection of mice against virulent virus infection by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture.

SummaryExperimental infection with HVJ (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) in mice was studied. Aerosol infection of newborn mice with the wild-type virus (HVJ-W) retarded the development of body weight and killed the animals within a few weeks. Large amounts of virus were isolated from both the lungs and the nasal turbinates of infected mice. In contrast, newborn mice exposed by inhalation to a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture showed no clinical signs and grew equally well as mock-infected animals. No infectious virus could be recovered from the lungs although thets mutant grew to moderate titre in the nasal turbinates.The prior inoculation of newborn mice with thets mutant virus induced a state of significant resistance to subsequent challenge with the virulent wild-type virus.No replication of challenge virus in both lungs and nasal turbinates could be detected and the animals were protected a lethal infection. It is suggested that an avirulent temperature-sensitive mutant which has lost the capacity to replicate in the lower respiratory tract but is still capable of multiplying in the nasal turbinates may be a promising candidate for use in live vaccines especially against the infectious disease of the lower respiratory tract.

Mechanism of interference between influenza A/WSN and B/Kanagawa viruses.

Simultaneous infection of MDCK cells with influenza viruses A/WSN and B/Kanagawa resulted in mutual interference with virus protein synthesis and in significant suppression of A/WSN growth. When infection by one virus preceded the other by 1 or 2 h, growth of the superinfecting virus was selectively inhibited at the level of transcription. Interference by the pre-infecting virus was strongly dependent on the expression of the viral genome but not on haemagglutinin activity. When the replication of both virus types was restricted to primary transcription by cycloheximide, the only translation products following removal of the drug were those of the preinfecting virus. This result was not affected by blocking secondary transcription by actinomycin D. These findings suggest that intertypic interference occurs at the level of primary transcription. This concept was supported further by the observation that a ts mutant of A/WSN (ts-65) with a defect in primary transcription interfered only with superinfection by B/Kanagawa at the permissive temperature.

Role of viral glycoproteins in haemolysis by influenza B virus.

Influenza B virus exhibited haemolytic activity at low pH, but the pH profile for its activity varied from strain to strain. The results of selective heat-inactivation of the enzyme activity of neuraminidase (NA) or elimination from virions of the enzyme-active portion of the NA molecule by trypsin digestion, suggest that proteolytically cleaved haemagglutinin, but not enzyme activity of NA, is essential for haemolysis by influenza B virus.

Characterization of Altered BHK Cells Resistant to HVJ (Sendai Virus) Infection

An altered baby hamster kidney cell culture which resists the c.p.e. of HVJ (haemagglutinating virus of Japan - the Sendai strain of parainfluenza I virus) has been obtained and characterized. These cells, designated BHK-R, were originally obtained by prolonged cultivation of cells surviving HVJ infection; they have been subcultured in the presence of HVJ. No infectious virus was recovered from BHK-R cells and no evidence for the presence of HVJ antigens in the cells was demonstrated by immunofluorescent staining. When BHK-R cells were inoculated with HVJ the growth of challenge virus was suppressed and no obvious cytopathic changes were detected, while these cells normally supported the replication of mumps, influenza, Newcastle disease, vesicular stomatitis or Sindbis viruses. BHK-R cells became susceptible to HVJ infection after serial subculture in growth medium free of HVJ. It was suggested that sialic acid residues present in the surface of BHK-R cell membranes and responsible for adsorption of HVJ were split off by the action of neuraminidase of virus particles, resulting in inhibition of the attachment of challenge virus of HVJ.

Effect of high salt treatment on influenza B viral protein synthesis in MDCK cells.

Based on the information that high salt inhibits the initiation of cellular mRNA translation which depends on the function of the 5′‐terminal structure of mRNA, we compared the effect of high salt on translation of host cellular mRNAs and influenza viral mRNAs, both of which are of 5′‐terminal structure. Brief exposure of influenza B virus‐infected MDCK cells to high salt medium resulted in a dose‐dependent inhibition of viral polypeptide synthesis as well as of cellular polypeptide synthesis, but it had less effect on synthesis of viral polypeptides, particularly nonstructural protein (NS). Under these conditions the Na+ content of the infected cells was significantly increased. A similar salt effect on in vitro translation of viral and cellular mRNAs extracted from infected cells was also observed. There was no significant difference in sensitivity to hypertonic block of in vivo translation of influenza viral mRNAs and vesicular stomatitis virus mRNAs, the latter of which possess a virus‐directed structure at the 5′‐terminus.

Plaque Formation by Influenza B Virus in a Porcine Kidney Cell Line

Proteolytic cleavage of hemagglutinin (HA) polypeptide to HAl, and HA2 enhances the infectivity of influenza A viruses without affecting specific hemagglutinating activity, allowing multiple cycles of growth of or plaque formation by the viruses (3,4). This is also the case with influenza B viruses. Growth of influenza B virus and plaque formation by the virus in cell cultures such as chick fibroblasts (CEF) (8, 10) or the HKCC line of hamster kidney cells (5) has been observed in the presence of proteolytic enzymes. Tobita (9) found highly efficient plaque formation by influenza B viruses in MDCK cells without proteolytic enzymes. Currently MDCK cells seem to be the most useful for biological, biochemical, and genetic studies of influenza B viruses. However, the disadvantage of using MDCK cells for plaque assay of the viruses is that plaques have to be counted soon after staining with neutral red since it is difficult to keep plaques clear after the addition of neutral red. This communication reports that the. ESK line of porcine kidney cells can serve for the plaque assay of influenza B virus. Influenza B/Kanagawa/73 strain virus was grown in the allantoic cavity of lO-day chick embryos at 34 C for 2 days and the harvested fluids were used as virus material. The ESK cell line was kindly supplied by Dr. T. Kumagai, The National Institute of Animal Health, Japan. The cells were dispersed by versine and grown in Eagles minimum essential medium (MEM) containing 10% calf serum. Monolayers of ESK cells in 4-cm petri dishes were inoculated with 0.1 ml of virus diluted with MEM containing 0.2% bovine serum albumin (BSA). After a I-hr adsorption period, 4 ml of MEM containing 0.6% noble agar and 0.2% BSA was added without removal of the inoculum. After incubation at 34 C for 3 days, 2 ml of agar medium containing 0.01 % neutral red was added. The plates were examined for plaques 24 hr later. The average plaque size was 2 mm in diameter. Further incubation did not usually produce a significant increase in the number of plaques, although there was an increase in plaque size (4 mm in diameter) (Fig. 1). Antiserum [hemagglutination-inhibition (HI) titer 1:5120] obtained from rabbits immunized with egg-grown Kanagawa strain completely inhibited the plaque formation when it was added to agar medium at a dilution of 1:400. It should be noted that ESK cells could be maintained in a viable condition for 3 days in the

Identification of DNA polymerase(s) involved in the repair of viral and cellular DNA in herpes simplex virus type 2-infected cells.

When human embryonic fibroblasts (HEF) were infected with herpes simplex virus type 2 (HSV-2), replicative viral DNA synthesis and some repair synthesis of cellular DNA were induced at the early stage of infection, but almost all DNA synthesis at the late stage of infection was derived from repair synthesis of cellular and viral DNA (Y. Nishiyama and F. Rapp, Virology 110, 466-475, 1981). In this study, we have assessed the effects of DNA polymerase inhibitors on repair DNA synthesis HSV-2-infected HEF. Both viral and cellular DNA syntheses during the late stage of infection were extremely resistant to aphidicolin and phosphonoacetic acid but partially sensitive to high concentrations of 1-beta-D-arabinofuranosylcytosine, while replicative viral DNA synthesis during the early stage of infection was very sensitive to all of those inhibitors. The results suggest that neither HSV-induced DNA polymerase nor cellular DNA polymerase alpha was involved in the repair synthesis of viral and cellular DNA but that cellular DNA polymerase beta was.

Lack of detectable DNA damage in cells productively infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) has many similarities in structural and biological properties to herpes simplex virus (HSV) , another human virus of the herpes group, but unlike HSV, HCMV is a relatively slow replicating virus and does not shut off host protein synthesis. Even during productive infection, HCMV stimulates various cellular functions including cellular DNA, RNA, and protein synthesis (2,5,6). However, little is known about the reason for the differences between HCMV and HSV in virus-cell relationships. Recent studies have shown that repair replication of cellular and viral DNA is induced during the early phase of infection in HSV-2-infected cells and that cellular DNA of the infected cells suffers extensive strand breaks (3). In the present study, we investigated whether HCMV induces DNA damage in productively infected cells. Human embryonic fibroblasts (HEF) of passage 5 to 20 were grown in Eagles minimum essential medium (MEM) supplemented with 10% fetal calf serum (fcs), 100 units/ml of penicillin, and 100 ftg/ml of streptomycin. HSV type 2 strain 186 and HCMV strain ADl69 were propagated in HEF by inoculating at low multiplicities (0.01-0.1 PFU/cell) (4). For UV-irradiation of cells, the medium was removed and cells were washed with phosphate buffered saline (PBS) and then irradiated for 20 sec at a dose of 20 ergs/mm2/sec as measured by a BlackRay ultraviolet dosimeter, model J-225. [MethyI-3H]thymidine (20.0 Ci/mmol) and [methyP4C]thymidine (51.0 mCi/mmol) were purchased from the New England Nuclear Corp., Boston. DNA analysis was performed by CsCI density gradient equilibrium centrifugation and by alkaline sucrose gradient sedimentation as described previously (3,4). Briefly, radiolabeled cells were harvested and digested for 6 hr at 37 C in a mixture of 0.25% Sarkocyl, 0.05 MEDTA and 0.5% heat-inactivated pronase. Radioactive material was then centrifuged in CsCI solution (density: 1.701 g/cm3) in 0.1 xSSC (0.015 M sodium chloride and 0.0015 M sodium citrate, pH 7.3) at 35,000 rpm for 55 hr using a Beckman 50.3 fixedangle rotor. 14C-Labeled cellular and HSV-2 DNAs containing no BUdR were used as markers. After centrifugation, fractions (about 0.09 ml) were collected

Analysis of the Inhibitory Effect of Canavanine on the Replication of Influenza RI/5+ Virus II. Interaction of M Protein with the Plasma Membrane

When influenza A/RI/5+ virus‐infected cells were incubated in medium to which 2 μg of canavanine (arginine analog) per ml had been added 4 hr after infection, all viral polypeptides were synthesized but the budding‐like process with the appearance of extracellular virus was completely inhibited. The plasma membrane isolated from these cells contained exclusively hemagglutinin (HA), and membrane (M) protein and nucleoprotein (NP) appeared to be associated with the nucleus, in contrast to untreated cells whose plasma membrane contained abundant HA, M protein, and NP. Disruption of canavanine‐treated cells by freeze‐thawing generated a number of hemagglutinating membranous vesicles or fragments containing exclusively HA. By isotope labeling it was found that the M protein synthesized in the presence of canavanine, together with HA and NP, is a canavanine‐substituted polypeptide. It is suggested that canavanine inhibits the formation of the mature envelope of influenza RI/5+, because of the inability of M protein to associate with the plasma membrane.