Phenotypic Mixing Research Articles

Phenotypic mixing between avian and mammalian RNA Tumor viruses: I. envelope pseudotypes of rous sarcoma virus

Pseudotypes of Rous sarcoma virus (RSV) with the envelope properties of six strains of mammalian leukemia viruses (MaLV) were produced after mixed infection in avian cells permissive for the replication of both types of virus. RSV pseudotypes with the envelope antigens of xenotropic and ecotropic murine leukemia virus were also obtained by fusion of mammalian cells producing leukemia virus with avian cells producing RSV, but RSV(MaLV) pseudotypes were not obtained by MaLV superinfection of RSV-transformed mammalian nonproducer cells. Functional RSV(MaLV) pseudotypes were obtained with both nondefective RSV and RSV defective in envelope antigens, but were not obtained with RSV which is defective in RNA-directed DNA polymerase.

The use of vesicular stomatitis virus pseudotype production in the study of a temperature-sensitive murine leukemia virus

A mutant of Moloney murine leukemia virus (MoLV), designated ts 3 was recently shown to have a temperature-sensitive defect associated with the release of mature virus particles budding from the cell membrane [Wong, P. K. Y., and McCarter, J. A., Virology 58, 396–408 (1974)] . In an attempt to determine whether the defective function resides in an envelope component of the virion, the formation of pseudotypes between VSV and ts 3 was studied under nonpermissive and permissive conditions of ts 3 infection. Whereas similar levels of phenotypic mixing were observed between VSV and wild-type MoLV at both 39 and 34°, the level of pseudotypes formed between VSV and ts 3 was found to be considerably lower at 39° (nonpermissive temperature) than at 34° (permissive temperature). The results of temperature-shift experiments indicate that two separate blocks to VSV ts 3 pseudotype production may occur depending on the length of time ts 3-infected cells are incubated at the nonpermissive temperature. Preincubation of ts 3-infected cells for 24 hr at 39°, prior to superinfection with VSV at 39°, prevents pseudotype formation. In contrast, brief incubation at 39°C, coincident with VSV infection, introduces a reversible block on the release of VSV (ts 3) pseudotypes from the cell membrane. Complementation of ts 3 through ts 3 (VSV) pseudotype production was not detected at the nonpermissive temperature.

Biological and electron microscopic studies on the phenotypic mixing of the thermolabile mutant of vesicular stomatitis virus,tl-17, with avian RNA tumor viruses

A thermolabile as well as thermosensitive mutant of vesicular stomatitis virus, VSV-tl-17, could be thermostabilized by phenotypic mixing with avian RNA tumor viruses (ATV). Biological, immunological and morphological studies revealed that this effect is due to a replacement of the thermolabile projections of the VSV by the avian tumor viral projections. The arrangement of projections of VSV and ATV on phenotypically mixed viria was studied by electron microscopy. A-type particles were detected in chick embryo cells which were doubly infected by ATV and VSV. Their intracytoplasmic appearance seemed to be the result of a disturbed maturation of ATV due to the relative deficiency of envelope proteins which were depleted by VSV maturation in the course of phenotypic mixing.

Phenotypic mixing of vesicular stomatitis virus and D‐type oncornavirus

After mixed infection of cells with vesicular stomatitis virus (VSV, thermolabile mutant tl 17) and oncornavirus type D, phenotypically mixed virions are formed containing the VSV genome and oncornavirus and VSV envelope. The virions are thermostable and have serologic characteristics of both viruses.

Viral pseudotypes and phenotypic mixing.

Viral pseudotypes and phenotypic mixing.

Phenotypic mixing between N- and B-tropic murine leukemia viruses: Infectious particles with dual sensitivity to Fv-1 restriction

In an effort to understand how N or B tropism is determined in murine leukemia virus (MuLV) particles, we analyzed the MuLV produced after dual infection of mouse cells by N- and B-tropic MuLV. The progeny MuLV from such a mixed infection are sensitive to Fv-1 restriction in both N- and B-type cells, but are still highly infectious for mouse cells which do not exhibit Fv-1 restriction. This dual sensitivity to Fv-1 restriction is a phenotypic property of MuLV produced by mixedly infected cells, since individual virus clones derived from this MuLV are either N- or B-tropic. In further experiments, we superinfected murine sarcoma virus (MSV)-transformed cells with mixtures of N- and B-tropic MuLVs. The rescued MSV is restricted in its ability to transform both N- and B-type cells. The results suggest that N- and B-tropic MuLVs specify different determinants, which are incorporated into virions along with the viral genome and which are the recognition sites for Fv-1 restriction. The presence of a given determinant in a virion renders the virus sensitive to restriction in cells of the opposite Fv-1 type.

Structural Interactions Between Viruses as a Consequence of Mixed Infections

Publisher Summary Structural interactions among viruses can occur in double infections. There are two pools of viral components in doubly infected cells and, unless virus maturation is highly specific, there is the possibility for structural interactions during maturation, leading to the production of particles made up from the structural determinants of both viruses. For a simple nonenveloped virus, the incorporation of the genome of one virus in a capsid is made up entirely of the protein subunits of the other virus. This kind of interaction is termed as “genomic masking.” A second class of particles structurally distinct from either of the interacting viruses would result from the enclosure of a single genome (or nucleocapsid) of either virus in a capsid (or envelope) assembled from protein subunits (or determinants) of both viruses. This phenomenon is known as “phenotypic mixing.” Phenotypic mixing may be almost a predictable outcome among structurally similar simple viruses, so long as cells become doubly infected and replication cycles overlap. Genomic masking is the only outcome among structurally dissimilar simple viruses. Such breakdown in the specificity of homologous assembly in double infections probably depends on the right combination and proportion of heterologous virus components in the right environment, which need not be the ideal environment for homologous assembly.

Sexual reproduction and parent-offspring conflict in the RNA “Tumour” virus/host relationship: Implications for vertebrate oncogene evolution

The following aspects of RNA tumour virus biology are stressed: (a) their relationship to transformation, i.e. their presence may cause transformation almost immediately, influence it probabalistically, be associated or not associated with transformation; (b) transmission may be horizontal or vertical (genetic or congenital); (c) viral interaction may lead to interference or genotypic or phenotypic mixing. The oncogene and protovirus hypotheses are discussed. Bearing the above in mind the following suggestions are made. (A) virus/host interaction (a) Those RNA viruses able to induce almost immediate transformation are pathogens. (b) Other types may perhaps be regarded as host genes able to alternate between the integrated and autonomous states. (c) Host alleles controlling these latter viruses have the characteristics of a complex mating control system, influencing the entry (and therefore recombination and conversion into the autonomous state) of certain groups of genes. Such recombination may sometimes be interspecific. (B) oncogene expression (a) Age and sex dependent oncogene expression occurs in periods of low “host” viability i.e. when transformation has a small effect on “host” fitness. (b) These periods are associated with immune system defects. (C) oncogene evolution Considerations in the evolution of a vertebrate oncogene may include:— (a) Transformation may be controlled by an altruistic “death allele”, active in young offspring of low viability, this allele saves parental care. (b) Genetically modified cells may be regarded as sexually reproduced progeny. Somatic and germ cell offspring are selected to extract the maximum possible parental investment. The reproductive success obtained from parental care is devalued by the effect on relatives (CR) and their degree of relatedness (Ro). New genetic material and low parent fitness reduces Ro and CR respectively. Any agent (chemicals, radiation or viruses) which reduces Ro or CR may be oncogenic without carrying oncogenes . (c) Transformation may increase the probability of horizontal transmission of that cells genotype and may be regarded as requiring parental investment. (d) Parent/offspring conflict (in the division of investment) predicts that the parent cells will “disagree” with the offspring cell as to when transformation should occur. (e) The oncogene is a vertebrate gene, the preceding points closely resemble the protovirus hypothesis except that conditions for gene expression (and not the gene itself) arise anew by recombination, mutation and ageing in each new generation.

Specificity of protein-RNA and protein-protein interaction upon assembly of TMV in vivo and in vitro

The possibility of genomic masking and phenotypic mixing was studied in vivo (in plants mixedly infected by two TMV strains). The combinations of strains were: (1) vulgare and U2, (2) temperature-sensitive coat protein mutant Ni118 and U2. In the experiments on mixed reconstitution the conditions of TMV maturation were modeled in vitro using combinations of RNAs and proteins from the same strains. Coating of viral RNA by the coat protein of a heterologous strain (termed genomic masking in vivo and mixed particle production in vitro) could be definitely demonstrated only when a doubly infected plant (or reconstituted mixture, respectively) contained two types of viral RNA and only one type of active protein (the protein of the second, temperature-sensitive strain was inactive at 33°). On the other hand, the genomic masking and mixed-particle production was absent or very limited (within the limitation of the assay) when RNAs and functional proteins of both strains were present in a mixedly infected plant ( in vivo) or in a reconstitution mixture ( in vitro). Thus, a high specificity of protein-RNA interaction exists when both RNA and protein have access to the homologous component upon TMV assembly. The absence of phenotypically mixed particles with a mosaic capsid in doubly infected plants in vivo implies that the specificity of protein-protein interactions between subunits should also be high. However, some mixed coat protein virus with mosaic capsid could be reassembled in vitro.

Further evidence for the existence of a viral envelope protein defect in the Bryan high-titer strain of Rous sarcoma virus

Electron microscopy observations of purified Bryan high-titer Rous sarcoma virus (BH RSV) using the freeze-drying technique showed that progeny made in the absence of a helper virus lacked visible surface projections or spikes. Phenotypic mixing experiments employing BH RSV and a thermolabile mutant of vesicular stomatitis virus, tl 17, yielded no evidence of pseudotype formation. Since tl 17 is known to be defective for an envelope glycoprotein, the lack of successful phenotypic mixing with BH RSV is consistent with the observed absence of viral spikes.

Replication of lactic dehydrogenase virus and Sindbis virus in mouse peritoneal macrophages. Induction of interferon and phenotypic mixing

Mouse peritoneal macrophages stimulated by thioglycollate can proliferate in culture for over 2 wk. Sindbis virus replicates in these cells, but not in the initial exudate culture. Lactic dehydrogenase virus (LDV) also produces higher yields in the exponentially growing cells. Proliferating macrophages infected with LDV induce a factor that inhibits the replication of Sindbis virus. The factor was identified as interferon by its species specificity and its acid stability. Macrophages that have been in culture for only 24 hr do not produce interferon. Replication of Sindbis virus in LDV-infected macrophages leads to phenotypic mixing between the two virions. We have detected both pure pseudotypes—virions containing the RNA of Sindbis virus and the envelope of LDV—and particles containing the envelope proteins of both virions. Our results demonstrate that the restricted host range of LDV is not due to the inability of the virus to adsorb to cells other than macrophages.

Heat-stable variant of human adenovirus type 5: characterization and use in three-factor crosses.

A variant of human adenovirus type 5 which is heat stable (hs) in vitro has been isolated following three rounds of heat inactivation at 52 C. The variant is genetically stable, both through vegetative viral passage and through recombination into other genetic backgrounds, which suggests that it arises from a single mutation. Three-factor crosses, using this mutant in conjunction with previously described temperature-sensitive mutants, suggest the hs mutation lies near the left-hand end of the genetic map. The mutant has been used to demonstrate the production of reciprocal recombinants in two-factor crosses. The mutational lesion is unknown, but phenotypic mixing occurs in hs times hs-+ infections, which suggests that it lies in a gene specifying a viri on structural protein. Other biological parameters examined have shown no differences from the wild-type hs-+.

Phenotypic Mixing Test to Detect and Assay Avian Leukosis Viruses

A phenotypic mixing (PM) test for detecting and assaying avian leukosis viruses (ALV) of the A, B, C, and D subgroups is described. An ALV and Rous sarcoma virus RSV-0) are phenotypically mixed by co-cultivating on C/O (cells susceptible to all subgroups of ALV) cells for a certain period. Then the RSV with the new virus property is assayed on C/E cells (cells resistant to infection with subgroup E leukosis/sarcoma viruses). The test is relatively simple and rapid, and its results are unequivocal. It is as sensitive as the more lengthy complement-fixation test (COFAL). The system is suitable for detecting avian leukosis viruses in samples such as heparinized blood, plasma, and embryo extracts.

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.

Characteristics of Infection by RSV

The duration of the latent period of RSV was studied in unsynchronized chicken cells using two methods. First, RAV1-producing cells were infected with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV;subgroup D), and new virions of SR-RSV (subgroup A) produced after phenotypic mixing with RAV1 were neutralized with an antiserum to RAV1. Secondly, uninfected chicken cells were infected with SR-RSV (D) in the presence of either arabionsylcytosine, actinomycin D or cycloheximide, to inhibit the virus production. In both methods, the early decrease of the virus titer showed that new virions were produced as early as 45 min after the beginning of the infection. Earlier times were not explored.

Products of complementation between temperature-sensitive mutants of simian virus 40

Temperature-sensitive mutants of the D complementation group of simian virus 40 exhibit delayed complementation. Analysis of the thermal stability, kinetic profiles in temperature shift experiments, and progeny of complementation have led to the hypothesis that delayed complementation is not true complementation, but the result of a very low level of leakiness, followed by phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants at the restrictive temperature.

Pseudotype formation between enveloped RNA and DNA viruses.

ENVELOPED RNA viruses from different groups participate readily with each other in phenotypic mixing during maturation at the plasma membrane of host cells. These ‘pseudotypes’ with the envelope antigens of one virus and the genome of the other have been demonstrated for simian virus 5 and vesicular stomatitis virus (VSV)1, for avian or murine RNA tumour viruses and VSV2–4, and for fowl plague virus and VSV5. (An exception has been the lack of interaction between Sindbis virus and VSV6.) We have now found virions containing the RNA genomes of VSV in the envelope antigens of a DNA virus, herpes simplex virus (HSV). These interactions between genomes and antigens of unrelated groups may be part of a mechanism for viral pathogenesis and oncogenesis.

Interactions between the vegetative states of phages lambda and T1.

Bacteria containing phage lambda in the vegetative state were produced either by induction of lambda lysogens or by infection of sensitive cells with lambda. These cells were superinfected with T1, and assayed for the production of lambda, T1, or both. Although most of the cells produced only lambda or T1, approximately 10% of the infectious centers were dual yielders. Examination of the progeny phage produced by the population of mixedly-infected cells showed that there was little, if any, phenotypic mixing, as determined by adsorption phenotype. T1am mutants in a variety of T1 genes were tested for their ability to exclude lambda, but none were defective in this ability. One gene of T1, gene 4, can be complemented by lambda.

Tumor induction by murine sarcoma virus in AKR and C58 mice. Reduction of tumor regression associated with appearance of Gross leukemia virus pseudotypes.

Adult AKR and C58 mice injected intramuscularly with murine sarcoma virus, Moloney isolate (M-MSV), developed high incidence of nonregressing local tumors. Histologically, these tumors revealed the typical pleomorphism of M-MSV sarcomas; in some cases, however, neoplastic tissue showed a nodular or diffuse growth of monomorphic myoblastlike cells, reminiscent of clonal aggregates. No depression of immune reactivity was found in M-MSV-injected mice as evaluated by direct hemolytic plaque-forming cells against SRBC and by virus-neutralizing antibody production. The MSV recovered from the induced tumors proved to be, by neutralization assay, a Gross (G)-MSV pseudotype. Moreover, tumor cell suspensions absorbed out cytotoxic antibody directed against G-cell surface antigens. Therefore, the conclusion was drawn that MSV with envelope characteristics of endogenous G leukemia virus had formed in vivo through a phenotypic mixing phenomenon. The failure of tumors to regress has been interpreted as mainly due to the partial unresponsiveness of host immune reactivity towards G-MuLV specified antigens. Since MSV-tumors arose in AKR mice after a very long latent period, the possibility was considered that this relative resistance might depend on immunologic mechanisms. In fact, M-MSV-injected AKR mice immunodepressed by goat antimouse lymphocyte serum or rendered partially tolerant by neonatal M-MuLV inoculation developed sarcomas with higher incidence and with a shorter latency. Furthermore, the MSV recovered from these early tumors proved to be the original Moloney pseudotype.

Evidence against phenotypic mixing between bacteriophage T4 wild type and T4v minus.

In a recent publication Shames et al. (1973) concluded that the UV-specific T4 endonuclease (a repair enzyme coded for by the gene v of wild-type T4) is a component of extracellular phage, which is injected into the host cell and can perform an early repair step without requiring gene expression. This notion is, however, not supported by results presented in this paper. Lysates obtained from mixed multiple infection of Escherichia coli cells with T4v(1) (-) and T4v(+) (or T4v(2) (-) and T4v(+)) failed to show the expected phenotypic mixing, i.e., incorporation of UV endonuclease into capsids of v(-) phages resulting in recognizable repair. The fraction of v(+) and v(-) particles in such lysates was determined by single-plaque analysis before and after irradiation with a UV dose at which virtually all survivors are particles having undergone repair. Even though our mixed infection conditions were most favorable for the possible occurrence of phenotypic mixing, none out of several hundred individual plaques from survivors were found to be genotypically v(-), whereas 30 were expected in the case that phenotypically mixed v(-) particles were repaired like T4v(+). Our failure to observe phenotypic mixing suggests that the data by Shames et al. reflect intracellular synthesis of endonuclease after phage infection.