Kenneth K. Takemoto

Virus-polysaccharide interactions: I. An agar polysaccharide determining plaque morphology of EMC virus

Abstract Wild-type EMC virus produced minute plaques (1 mm at 4 days) on L cell monolayers with an occasional mutant which produced large plaques (8–10 mm at 4 days). A sulfated polysaccharide present in agar inhibited the growth of the minute-plaque former, but had no effect on the large-plaque mutant. In the absence of the agar polysaccharide, the minute-plaque former actually multiplied more rapidly than the mutant. Two other macroanions, polyglucose-SO 4 and heparin, were also inhibitory to the virus that formed small plaques.

Virus-cell relationship in a carrier culture of HeLa cells and Coxsackie A9 virus.

Abstract Although relatively resistant to infection with Coxsackie A9 virus of monkey kidney (MK) culture origin, HeLa cells have been found capable of supporting growth of this virus. When heavy inocula of the A9 virus were used in cultures of HeLa cells adapted to growth in medium containing 10% human, horse, or calf serum, the cultures showed viral cytopathogenic effects (CPE) but recovered after addition of fresh growth medium. These cultures could be passed serially for many cell generations with consistent recovery of the virus in the supernate. The term “carrier culture” has been used to designate this type of virus-cell relationship ( Lwoff, 1953 ). No evidence for a lysogenic state was found in this system. Animal or human immune serum eliminated virus from the cultures and clonal isolates of cells from the carrier culture were negative for virus. Only a small proportion of the cells in carrier cultures were infected. Prolonged cultivation of the carrier cultures tended to select not only cells with increased resistance to Coxsackie A9 infection, but also virus with altered properties. Virus from carrier cultures differed from the original virus grown in MK by its (a) lack of virulence for suckling mice, (b) increased virulence for HeLa cells, (c) slow plaque formation in MK plaque plates, and (d) antigenic composition. The evidence presented indicates that the principal factors responsible for continued maintenance of the carrier cultures are the relative insusceptibility of HeLa cells to Coxsackie A9 virus, especially in the state of cellular activity present in growth medium, and the prevention of high multiplicities of virus in the cultures by rapid thermal inactivation.

The effect of interferon on SV40 T antigen production in SV40-transformed cells

Abstract Continuous passage in the presence of interferon failed to reduce the content of SV40 T antigen in a line of SV40-transformed mouse cells (3T3). However, marked inhibition of SV40 T antigen formation resulted when normal 3T3 cells were pretreated with interferon and subsequently infected with SV40 virus. The implications of the undiminished translation of viral genetic information in transformed cells in which an interferon-induced antiviral state exists are discussed. It is suggested that SV40-transformed cells produce molecules of messenger RNA (mRNA) containing both host and viral information, and that the presence of some host genetic information on the mRNA molecule renders it insensitive to the interferon system.

Electron microscopic observations on multiple polyoma virus-related particles

Abstract Isopycnic banding of small-plaque polyoma virus in CsCl produced four bands at densities 1.28, 1.29, 1.33, and 1.34. Examination of these fractions in the electron microscope revealed the upper band to consist of 48 mμ shells incompletely filled with PTA. These were interpreted as being 48 mμ shells with an internal 38 mμ shell. The second band consisted of three sizes of separated, capsomered shells 48, 38, and 22 mμ in diameter which are believed to consist of 72, 32, and 12 “capsomeres”, respectively. The lower two bands were not morphologically different, consisting of complete virus particles. A 26 mμ shell of DNA was shown in complete particles by uranyl acetate staining, and a 38 mμ capsomere shell was demonstrated in situ between the 26 mμ DNA shell and a ring of residual stain between the 48 and 38 mμ shells. These observations suggest that the polyoma virus may be constructed (from interior to exterior) of a 22 mμ shell of capsomeres, a thin shell of DNA, a 38 mμ shell of capsomeres and a 48 mμ protein shell. A fifth layer of phospholipid covers some particles to a diameter of about 60 mμ A similar picture of three sizes of spherical particles was seen with SV40 virus. Filamentous, capsomered forms were found in large number only in KBr “cushion” fractions; apparently filaments were destroyed by CsCl. Filaments also occurred in three general sizes, 22, 38, and 48 mμ Possible roles of filamentous forms in virus replication are discussed.

Replication of polyoma virus in mouse embryo cells: Electron microscopic observations

Abstract Single step replication of polyoma virus in mouse embryo (ME) cells has been examined by electron microscopy. Cells phagocytose individual virus particles or membrane bound aggregates into the cytoplasm. Individual particles appear in the cytoplasm as 50–60 mμ membrane-bounded viruses. Either 38 mμ or 50–60 mμ particles may be found in the larger phagocytic inclusions. As early as the eighth hour of infection, virus particles have been occasionally observed between the two nuclear membranes; however, at later periods they are frequently observed in this location. By the twentieth hour of infection, small, densely staining, bundles of filaments are observed in multiple loci in nuclei. These bundles increase in number with time and are often surrounded by typical 38 mμ “nuclear” virus particles which may form intranuclear crystals as early as 24 hours after infection. Virus in cells whose nuclei are degenerating exhibit a remarkable affinity for nuclear, cytoplasmic and cell surface membranes. These observations support the suggestion of Bernard et al. (1959) that the nuclear filament is a precursor of polyoma virus.

THE BASIS FOR THE SIZE DIFFERENCES IN PLAQUES PRODUCED BY VARIANTS OF ENCEPHALOMYOCARDITIS (EMC) VIRUS.

Abstract A sulfated polysaccharide in agar has been shown to be responsible for the size differences in the plaques produced by two variants of encephalomyocarditis (EMC) virus. Sulfated polysaccharides (using sodium dextran-SO 4 as a model compound) interfere with the adsorption of the wild-type ( r + ) virus, but not the large-plaque ( r ) variant. At a constant virus concentration, the effect of dextran-SO 4 is concentration dependent, and is also a function of pH and ionic strength. Probably an ionic interaction between sulfated polysaccharide and r + virus effectively binds or restricts the virus and thereby reduces the absolute number of plaque-forming units adsorbed to susceptible cells per unit time and reduces the plaque size in contrast to the r variant, which is not bound by the sulfated polymer in the agar overlay.

Inhibition of Herpes Virus by Natural and Synthetic Acid Polysaccharides

Summary The effect of 4 acid polysaccharides on 2 variants of herpes virus was investigated by 3 different methods for measuring anti-viral activity. Dextran sulfate inhibited adsorption, plaque formation, and multicyclic viral growth in liquid medium. Heparin and agar polysaccharide inhibited adsorption and virus multiplication in liquid medium, while chondroitin sulfate was completely ineffective as an inhibitor. No diffference was noted in the behavior of the 2 variants with respect to their sensitivity toward the 4 sulfated polysaccharides.

Recovery of SV40 virus with genetic markers of original inducing virus from SV40-transformed mouse cells.

Cells from three different mouse strains were transformed in vitro with 2 mutants of SV40 as well as with wild-type virus. SV40 virus could be recovered from transformed ALN and AKR cells by cocultivation with susceptible cells, but virus production occurred much sooner when cells were allowed to form heterokaryons with UV-inactivated Sendai virus. 3T3 transformed cells which did not yield virus by simple cocultivation did yield virus by the cell-fusion technique. SV40 virus recovered from transformed cells was examined and compared with the original transforming virus for three genetic markers: (a) plaque size in primary AGMK, (b) growth at 40°, and (c) plating efficiency and plaque size in an established AGMK cell line, CV-1. In all cases, the virus recovered possessed the markers of the original parental virus.

Evaluation of normal and neoplastic human tissue for BK virus

Abstract Despite an extensive search using sensitive DNA hybridization techniques, we were unable to demonstrate the presence of BK Virus DNA sequences in human neoplastic tissues. Evaluation of numerous human tumor cell lines for BK Virus T-antigen and sera from patients with known malignancies for anti-BK Virus T-antibody was also negative.

Sensitivity and resistance of type 1 polioviruses to an inhibitor in certain horse sera.

Abstract An inhibitor against type 1 poliovirus associated with the gamma globulin of certain horse sera was investigated. Addition of inhibitory horse serum (IHS) at a concentration of 2 1 2 % in the overlay medium caused marked reduction in plaque size and a delay of 24–48 hours in plaque development. Plaque counts were unaffected at this serum concentration, but were reduced by 50 % with 5 % serum. Inhibitor-resistant particles capable of forming normal-sized plaques in the presence of IHS were found in a plaque-purified pool of type 1 poliovirus (Mahoney strain). Based on plaque size, inhibitor-sensitive and -resistant particles could be differentiated in naturally occurring type 1 virus in stools of patients with paralytic poliomyelitis. The inhibitor had no effect on plaque development of type 2 and 3 polioviruses, Coxsackie A9, vaccinia, or encephalomyocarditis virus (EMC). The inhibitor differs from the previously described bovine serum inhibitor in not having neutralizing ability; no evidence was found that it is viral antibody. Growth-curve experiments showed that the inhibitor reduces rate of adsorption of virus to susceptible cells and delays release of virus from infected cells.