Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).     

Failure of defective interfering particles of Sindbis virus produced in BHK or chicken cells to affect viral replication in Aedes albopictus cells.

Whereas defective interfering particles of Sindbis virus are readily produced in BHK-21 cells or chicken embryo fibroblasts by the techniques of serial undiluted passage, similar methods failed to generate such particles in Aedes albopictus cell cultures. In addition, Sindbis virus stocks produced in BHK-21 cells or chicken embryo fibroblasts and which contained defective interfering particles, when tested in A. albopictus cells, failed (i) to interfere with the replication of standard Sindbis virus and (ii) to change the pattern of intracellular viral RNA synthesis from that produced by infection with standard Sindbis virus alone. We conclude that defective interfering particles of Sindbis virus generated in chicken or hamster cells are silent or inert in mosquito cells.     

Mosquito cells infected with vesicular stomatitis virus yield unsialylated virions of low infectivity.

Vesicular stomatitis virus propagated in and released from Aedes albopictus cells had the normal complement of viral proteins; the glycoprotein contained carbohydrate but no sialic acid. These virions had markedly reduced hemagglutinating activity and exhibited a very high ratio of physical particles to infectious virus. In vitro sialylation of vesicular stomatitis virions grown in mosquito cells resulted in a 100-fold increase in both infectivity and hemagglutination titers to levels approaching those of virus grown in BHK-21 cells. These experiments provide an example of host-controlled modification of viral infectivity.     

Preliminary Physiological Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.     

Novel antiviral activity found in the media of Sindbis virus-persistently infected mosquito (Aedes albopictus) cell cultures.

Aedes albopictus (mosquito) cells persistently infected with Sindbis virus for a period of 6 months release into the medium a low-molecular-weight material capable of specifically reducing the yields of Sindbis virus during the "acute phase" of infection in mosquito cells. The antiviral activity was produced in detectable levels at 3 days after infection, and its concentration in the extracellular medium increased thereafter. The antiviral activity was inactivated by treatment with the enzyme protease K and heat. It was not activated by treatment with antibody prepared against extracts of Sindbis virus-infected BHK-21 cells. The antiviral activity differs from interferon produced by vertebrate cells in that it is virus specific as well as cell specific.     

Morphology of BHK-21 Cells Infected with Sindbis Virus Temperature-Sensitive Mutants in Complementation Groups D and E

BHK-21 cells infected with temperature-sensitive mutants of Sindbis virus in complementation groups D and E differed in their appearance under nonpermissive conditions. Cells infected at nonpermissive temperature with virus defective in complementation group E had nucleocapsids attached in large numbers to the inside surface of the host plasma membrane. Infection with a group D mutant produced nucleocapsids that did not attach to the plasma membrane but rather remained free in the cell cytoplasm.     

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.     

Growth, plaque assay and immunofluorescent studies on Tataguine virus in cell culture.

The growth characteristics of Tataguine virus were studied in Cercopithecus monkey kidney (Vero); rhesus monkey kidney (LLC-MK2), baby hamster kidney (BHK-21); porcine kidney (PK-15), mouse fibroblasts (L-929) and Aedes albopictus cell monolayers. The virus replicated without producing any cytopathology in Vero, BHK-21 and Aedes albopictus: but not in the other three cell culture systems. Two or three subsequent serial blind passages in those cultures supporting the growth of the virus did not produce any appreciable increase in virus titre. Immunofluorescent staining of inoculated Vero cells demonstrated the presence of Tataguine virus antigen in the cytoplasm of infected cells. Plaques 1--1.5 mm in diameter were produced only in Vero cell culture. In neutralization tests performed on Tataguine virus, immune mouse and hamster sera, higher antibody titres were obtained by plaque reduction than mouse protection tests.     

Mutant identifying a third recombination group in a bunyavirus.

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.     

Temperature-Sensitive Virus from Aedes albopictus Cells Chronically Infected with Sindbis Virus

Cultures of Aedes albopictus cells persistently infected with wild-type Sindbis virus (SV-W) give rise to small plaque-forming mutants which are also temperature sensitive. These mutants, designated SV-C, are neutralized by antiserum produced against SV-W. Mutant ts clones were isolated from SV-C by plaque purification. After serial undiluted passage in BHK or mosquito cells, each of the clones gave rise to ts(+) revertants which, however, remained mutant with respect to plaque morphology. Nineteen of 20 clones derived from SV-C were RNA(+), and one was RNA(-) (SV-C-2). The RNA synthesizing activity, once induced in infected cells by SV-C-2, was stable at the nonpermissive temperature (39.5 C). All clones derived from SV-C were inactivated at 60 C much more quickly than was SV-W. It was not possible to demonstrate complementation between any of the SV-C clones.     

Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell.

The microviscosities of the hydrophobic regions of the membranes of intact Semliki forest and Sindbis viruses grown on BHK-21 cells, of liposomes derived from the extracted viral lipids, and of protease-treated virions were measured by fluorescence depolorization using the fluorescence probe 1, 6-diphenyl-1,3,5-hexatriene. The intact virus membranes were found to have a higher microviscosity than did virus-derived liposomes, indicating the viral envelope proteins contribute to microviscosity. However, protease-treated virus, devoid of protruding spikes but with residual lipophilic peptide tails, was found to have a microviscosity more similar to that of the intact virus than to that of protein-free liposomes. Sindbis virus grown in BHK-21 cells at 37 C had a much higher microviscosity than did Sindbis virus grown on Aedes albopicuts cells at 22 C. Sindbis virus grwon in A. albopictus and BHK-21 cells also gave higher microviscosity values than did the intact host cells. These data indicate that both the virion proteins and the cellular lipids selected during viral growth and maturation contribute to the increased microviscosity of togavirus membranes.     

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.     

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Phospholipid Synthesis in Sindbis Virus-Infected Cells

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.     

Resuscitating mutations in a furin cleavage-deficient mutant of the flavivirus tick-borne encephalitis virus

Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The "pr" part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37 degrees C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28 degrees C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.     

Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.     

Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo

Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E. The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)-dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.