Assembly of membrane glycoproteins studied by phenotypic mixing between mutants of vesicular stomatitis virus and retroviruses

Temperature sensitive (ts) mutations of vesicular stomatitis virus (VSV), Indiana serotype, which belong to complementation group V (tsV) have been shown to affect the viral envelope glycoprotein, or G protein. When ts V mutants are grown in cells producing avian leukosis viruses, the titers of infectious VSV obtained at the nonpermissive temperature are 10⁴-fold higher than in control cells. Cells releasing murine leukemia viruses or avian reticuloendotheliosis virus rescue VSV ts V mutants much less efficiently. The rescued virions have the properties of envelope pseudotypes in that their host range is restricted to that of the helper retrovirus, they are neutralized by anti-retrovirus antibodies but not anti-VSV antibodies, and they are not thermolabile. Sensitive serological techniques, including the use of complement-mediated virolysis, immunoprecipitation, and monoclonal antibody reacting with G protein, show that VSV pseudotypes produced at the nonpermissive temperature have no detectable G protein, whereas VSV particles released from retrovirus infected cells at the permissive temperature have mosaic envelopes bearing both VSV G protein and retrovirus glycoprotein. In mixed infections of Rous sarcoma virus (RSV) and VSV ts V mutants, pseudotype particles with RSV genomes and VSV envelope antigens are produced only at the permissive temperature. In contrast, substantial yields of RSV(VSV) pseudotypes but no VSV(RSV) pesudotypes are obtained at the nonpermissive temperature with VSV carrying mutations in complementation group III, which affect M protein. Thermolabile VSV tsV mutants form RSV(VSV) pseudotypes which also are thermolabile. The kinetics of heat inactivation of G protein function in tsV mutants is the same in VSV particles with unmixed envelopes and with mosaic envelopes. From these studies of phenotypic mixing we draw the following conclusions: (i) The synthesis of functional M protein but not G protein is essential for the maturation of VSV virions. (ii) VSV M protein is not required for the assembly of G protein into retrovirus virions. (iii) The thermolabile nature of tsV VSV mutants is an intrinsic property of the G protein, independent of the type of virion into which it is incorporated and of other viral glycoproteins which may be assembled into the envelope of the same virion.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) III. Involvement of SV40 gene A in the expression of TrAg in permissive cells.

Temperature-sensitive (ts) mutants of simian virus (SV40) which map in the early region of the SV40 genome were used to determine the role of the viral genome in the expression of SV40-specific transplantation rejection antigen (TrAg). The results indicated that tsA mutants (1612, 1637, 7, and 28) did not induce the expression of SV40-TrAg at the surface of infected permissive African green monkey kidney cells (TC-7) at 41° but did induce the expression of TrAg at the permissive temperature (33°) in TC-7 cells. Wild-type SV40 and late SV40 temperature-sensitive mutants (tsBC1602, tsBC1606, tsB8, and tsC219) induced SV40-TrAg in TC-7 cells at nonpermissive and permissive temperatures with equal efficiency. One of the mutants belonging to complementation group D (tsD1601) was defective in inducing SV40-TrAg at 41°. Kinetic studies indicated that SV40-TrAg appears by 18 hr after infection at 41° and 38 hr post-infection at 33°, paralleling closely the synthesis of T antigen. The synthesis of immunoreactive T antigen in TC-7 cells infected with tsA mutants at nonpermissive temperature did not correlate with the inability of tsA mutants to express TrAg at nonpermissive temperature. We conclude that the expression of TrAg in SV40-infected cells depends upon normal functioning of the A gene.     

Characterization of a temperature-sensitive mutant of frog virus 3 defective in DNA replication

We have characterized the defect of a temperature-sensitive (ts) DNA^? mutant (ts 6642) of frog virus 3 (FV 3). At the nonpermissive temperature (30°) ts 6642 synthesized <3% of the viral DNA that was synthesized at the permissive temperature (23°). When ts 6642-infected cells were incubated at 30° for 4.0 hr and then shifted to permissive temperature, viral DNA synthesis started immediately even when protein synthesis was inhibited at the time of shiftdown. This result implies that at 30°, ts 6642 synthesized all the proteins required for viral DNA replication but that one of these was nonfunctional at the nonpermissive temperature. Further characterization revealed that ts 6642 was probably defective in the initiation of DNA replication. This conclusion was based on the following data: When ts 6642-infected cells incubated at 23° for 4.0 hr were shifted to 30°, there was a gradual decrease in viral DNA synthesis. By 1 to 1.5 hr after the shiftup, viral DNA synthesis was completely inhibited. Analysis of the density of the DNA synthesized after a shiftup in the presence of BUdR and FUdR suggested that residual viral DNA synthesis represented chain elongation, and not initiation of new rounds of DNA replication. The defective protein was therefore involved in the initiation process. Both wild-type FV 3 (FV 3⁺) and ts 6642 induced the synthesis of thymidine kinase and DNA polymerase at 30°. Therefore, neither of these enzymes was involved in the DNA replication defect of ts 6642.At the nonpermissive temperature, ts 6642 synthesized all the viral proteins that were detectable at the permissive temperature. However, synthesis of late proteins was delayed, and never reached wild-type levels. Furthermore, the rate of synthesis of late proteins at 30° became dependent upon the multiplicity of infection. These results reinforce our previous conclusion (R. Goorha and A. Granoff, 1974, Virology60, 237–250) that in FV3⁺-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.     

ts Transformation mutants of avian sarcoma virus PRCII: lack of strict correlation between transforming ability and properties of the P105-associated kinase

Three mutants of avian sarcoma virus PRC-II, LA42, LA46, and LA47, have a temperature-sensitive (ts) lesion affecting cellular transformation in vitro. At the nonpermissive temperature (41.5°) they do not induce focus formation in fibroblast cultures. LA46 also fails to induce colonies in soft agar at 41.5°, while LA42 and LA47 have retained this ability. The mutations appear to be located in the transformation-specific insert of the defective sarcoma virus genomes, since association with different wild-type (wt) helper viruses does not lead to changes in the transforming phenotypes. The transformation-specific protein P105 of PRCII is detectable at the nonpermissive temperature in moderately reduced quantity in wt- and LA42-infected cells, while the amounts of P105 precipitable from LA47-infected cultures under these conditions are significantly decreased. LA46 made barely detectable quantities of P105 at 41.5°. This temperature sensitivity of LA46 in the synthesis of P105 may reflect the greatly reduced levels of transformation-specific RNA in LA46-infected cells at 41.5°. Intracellular phosphorylation of P105 was not found to be ts in the mutants or in wt PRCII at both serine and tyrosine acceptor sites. P105 extracted from wt-, ts mutant- or wt-revertant-infected cells at permissive and nonpermissive temperatures did not vary significantly in the specific activity of its associated protein kinase as assayed in vitro by phosphorylation of P105 itself. However, preincubation of P105 in vitro at 41.5° revealed greater instability of protein kinase reactions measured in P105 immunoprecipitates from mutant- as compared to wt-infected cells. Also the elevation of cellular phosphotyrosine, characteristics of PRCII-transformed cells, was greatly reduced in ts mutant-infected cells at the nonpermissive temperature but was restored to wt levels in genetic revertants derived from the ts mutants. These observations suggest that there is no direct correlation between in vivo or in vitro phosphorylation of P105 and the induction of all parameters of oncogenic transformation. The increase of total cellular phosphotyrosine appears to be correlated with focus formation, but not with the ability to induce agar colonies.     

A mouse embryo cell line carrying an inducible, temperature-sensitive, polyoma virus genome.

Secondary monolayer cultures from whole mouse embryos were infected at 39° with ts-P155, an early temperature-sensitive mutant of polyoma virus that transforms but replicates poorly at this temperature (W. Eckhart, 1969, Virology38, 120–125; W. Eckhart, 1974, Cold Spring Harbor Symp. Quant. Biol.39, 37–40). The cultures were then passaged at 39° in the presence of polyoma virus antiserum until sufficient cell degeneration occurred to allow detection of foci of cells exhibiting lack of contact-inhibition. From one such focus, a cell strain—designated Cyp—was derived and cloned at 39°. Whether cloned or uncloned, Cyp cells all seem to exhibit essentially the same phenotype. At 39°, they display several properties of transformed cells and, as indicated by immunofluorescence, synthesize polyoma T antigen. They do not produce any readily detectable amounts of viral DNA or V antigen and only low amounts of viral hemagglutinin at 39°, although they are still fully permissive for polyoma virus replication at this temperature. Upon transfer to 33°, Cyp cells undergo a massive cytopathic effect detectable within 30 to 34 hr and produce large amounts of infectious, temperature-sensitive polyoma virus. Viral DNA accumulating in Cyp cells after temperature shift-down consists primarily of covalently closed monomers and, upon cleavage by HpaII, yields fragments characteristic of the polyoma virus strain from which the ts-P155 mutant had been derived. When present in the medium during temperature shift-down, polyoma virus antiserum neither curtails the cytopathic effect nor depresses viral DNA synthesis, although it suppresses virus production. These observations are in agreement with the results of infectious center assays, suggesting that most Cyp cells produce virus following transfer to 33°.     

Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus.

Four DNA-negative temperature-sensitive (ts) mutants of human cytomegalovirus, belonging to different complementation groups, were studied for their ability to induce cell DNA synthesis and DNA polymerase in permissive human embryo lung (HEL) and nonpermissive rabbit lung (RL) cells. These is mutants stimulated host cell DNA synthesis in HEL and RL cells and DNA polymerase activity in HEL and RL cells at permissive (33.5°) and nonpermissive temperatures (39.5°). Salt stimulation of induced DNA polymerase activity was used to distinguish between virus and cell DNA polymerase from HEL cells. DNA polymerase activity was stimulated by 100 mM (NH₄)₂SO₄ at either 33.5 or 39.5° in cultures infected with three of the mutants (ts 9, is 153, and ts 155). However, DNA polymerase activity was not stimulated by 100 mM (NH₄)₂SO₄, in cultures infected with one of the ts mutants (ts 13). These data suggest that at least four cistrons control the synthesis of virus DNA and that virus DNA synthesis is not required for the induction of cell DNA synthesis.     

Induction of interferon by temperature-sensitive mutants of Newcastle disease virus

Spontaneously-selected and mutagen-induced temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were used to study interferon induction in chick embryo (CE) cells at temperatures permissive (37°) and nonpermissive (42°) for virus replication. Both infectious and UV-irradiated virus were tested for interferon-inducing ability in cells pretreated or not pretreated with homologous interferon. At 37°, only UV-irradiated NDV was capable of inducing interferon in cells not treated with interferon before infection. In cells pretreated with interferon, on the other hand, both unirradiated and UV-irradiated virus stimulated the production of interferon. At 42°, the interferon-inducing phenotype for some UV-irradiated ts mutants was dependent on whether or not cells were pretreated with interferon. For example, out of 10 mutants examined, one UV-irradiated ts mutant induced interferon in both untreated and interferon pretreated cells; 7 mutants failed to induce in untreated cells but induced from 25–100% of the wild-type level of interferon in cells pretreated with interferon; and two mutants failed to induce interferon in both types of cells. In addition, one mutant (NDV₀ts-100) induced low or undetectable levels of interferon at both 37° and 42°, conditions under which wild-type virus (NDV₀) produced significant levels of interferon. Co-infection of cells with UV-irradiated ts-100 and a preparation of NDV₀ exposed to prolonged irradiation resulted in considerable production of interferon. These results suggest the possibility that more than one virus function may be involved in interferon induction by NDV in CE cells.     

Synthesis of virus-specific polypaptides by temperature-sensitive mutants of herpes simplex virus type 1.

The polypeptide phenotypes of 22 temperature-sensitive (ts) mutants of herpes simplex virus type 1 were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis of mutant-infected cells at permissive and nonpermissive temperatures. Following analysis of isotopically labeled polypeptides synthesized from 4–24 hr postinfection, the mutants were divided into four major phenotypic groups which include: (1) DNA^?ts mutants which share several common polypeptide defects, (2) DNA^±ts mutants which exhibit polypeptide profiles resembling the DNA^?ts mutants, (3) DNA⁺ts mutants which exhibit polypeptide phenotypes differing only slightly from that observed in wild-type virus-infected cells grown at 39°, and (4) DNA⁺ts mutants which exhibit no detectable alterations in their polypeptide profiles when compared with that of the wildtype virus. When the polypeptide phenotypes of the mutants were compared with previously determined mutant characteristics, including synthesis of viral DNA, thymidine kinase, DNA polymerase, and physical virus particles, a correlation was consistently observed between mutant polypeptide and viral DNA phenotypes.     

Phage lambda DNA injection into Escherichia coli pel- mutants is restored by mutations in phage genes V or H.

Mouse cells in culture contain two distinct forms of thymidine kinase enzyme activities. These two enzymes have been separated by polyacrylamide gel electrophoresis into a 0.2 R_f and a 0.5 R_f activity. The 0.2 R_f enzyme was found only in actively growing cells, while the 0.5 R_f form of thymidine kinase is the mitochondrial-associated enzyme and is most prominent in resting cells in culture. SV40 infection of these resting cells results in an increased specific activity of only the 0.2 R_f form of this enzyme.SV40 wild-type and SV40 tsBC and tsC mutants stimulated the levels of the 0.2 R_f thymidine kinase in resting cells after viral infection at either the permissive temperature (32°) or the nonpermissive temperature (41°). Five different SV40 tsA mutants (tsA7, 28, 30, 58, and 209) and two different SV40 tsD mutants (tsD202, 270) only stimulated thymidine kinase activity at the permissive temperature. Little or no 0.2 R_f thymidine kinase activity could be detected in tsA or tsD mutant-infected cells at the nonpermissive temperature. The SV40 tsA255 mutant appeared to be an exception to the A mutant class in that it stimulated the 0.2 R_f thymidine kinase activity at both permissive and nonpermissive temperatures.These results indicate that the SV40 A gene product may be required directly, and the D gene product indirectly, in the stimulation of cellular enzyme activities following viral infection.     

Temperature-sensitive mutants of influenza virus: A mutation in the hemagglutinin gene

A mutant (ts-61S) belonging to a single recombination-complementation group (Group VI) was obtained by segregation of an influenza virus WSN (HON1) temperature-sensitive double mutant (ts-61) that possessed mutational lesions characteristic of Groups V and VI. The segregant retained the thermolabile hemagglutinating activity of the parental mutant, ts-61, but lost the defectiveness in virion RNA synthesis manifested by the parent at the nonpermissive temperature. No hemagglutinating activity developed in cells infected with ts-61S at the nonpermissive temperature. In rescue experiments all HO-serotype progeny from the cross between ts-61S (HO-serotype) and temperature-resistant H3-serotype virus were temperature-sensitive, localizing the ts defect in the hemagglutinin gene. No glycosylated hemagglutinin polypeptide was detected in the polyacrylamide gel electropherogram of cells infected with ts-61S at the nonpermissive temperature, whereas the synthesis of neuraminidase (the other virion glycoprotein) proceeded normally at both permissive and nonpermissive temperatures. The results indicate that the Group VI mutation is in the gene coding for the viral hemagglutinin.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) II. Isolation and characterization of additional temperature-sensitive mutants of SV40.

Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive (ts) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the tsA mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for tsBCll previously. Mutants in this group did not produce V antigen at high temperature.     

A mutant of Rous sarcoma virus with a conditional defect in the determinant(s) of viral host range.

A temperature-sensitive mutant of the Prague strain of Rous sarcoma virus of subgroup C, tsPH734PR-C, replicates much less efficiently at the nonpermissive (41°) than the permissive (35°) temperature while transforming equally well at both temperatures. In contrast, the wild-type parent, wtPR-C, is able to replicate and to transform chick embryo fibroblasts equally well at both 35° and 41°. Two lines of evidence suggest that tsPH734PR-C is defective in the synthesis or utilization of virus envelope glycoproteins GP85 and GP35. First, tsPH734PR-C appears to be defective in the incorporation of the virus envelope glycoproteins GP85 and GP35 into the noninfectious virus particles synthesized at 41°. Second, tsPH734PR-C, a host range subgroup C virus, is not complemented for replication of subgroup C virus at 41° by coinfection of cells with RAV-6 avian leukosis virus of subgroup B or by coinfection with the defective Bryan strain of Rous sarcoma virus, BH-RSV(?).     

Analysis on the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B77

Ts 334, a temperature sensitive mutant of avian sarcoma virus B77, cannot produce infectious progeny nor induce neoplastic transformation at the nonpermissive temperature. In order to clarify the relationship between these two functions of ts 334 we attempted to (1) isolate and characterize nonconditional transformation defective (td) mutants from ts 334, (2) isolate and characterize recombinants between ts 334 and RAV-1, and (3) reexamine rescue of ts 334 with RAV-1 at the nonpermissive temperature.All seven nonconditional td mutants isolated from ts 334 kept their temperature sensitive character in replication, although they had lost transforming capacity both at the permissive and at the nonpermissive temperatures. They appear to have a temperature sensitive step in virus maturation like ts 334.The helper function of these td mutants for the defective Bryan high titer strain of Rous sarcoma virus is also temperature-dependent.Two recombinants were isolated from cells coinfected with ts 334 and RAV-1. These recombinants combined the cell-transforming ability of ts 334 and of the envelope properties of RAV-1. These two recombinants were unable to induce cell-transformation but grew well at the nonpermissive temperature.In RAV-1 producing cells not only the genome of ts 334, but also the envelope property of ts 334 were rescued at the nonpermissive temperature, though cell transformation was not observed.These observations suggest that ts 334 has two mutations, one affecting reproduction and another cell-transformation capacities.     

Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus.

Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.     

Pseudotypes of avian sarcoma viruses with the envelope properties of vesicular stomatitis virus.

Upon superinfection of cells producing Rous sarcoma virus (RSV) with temperature-sensitive mutants of vesicular stomatitis virus (VSV), two kinds of pseudotype viruses are produced: VSV genomes within particles bearing the envelope antigens of RSV, denoted VSV(RSV), and RSV genomes within particles bearing the envelope antigens of VSV, denoted RSV(VSV). The VSV(RSV) pseudotypes are recognized as the fraction of plaque-forming units resistant to neutralization by antiserum to VSV or, in the case of thermolabile envelope mutants of VSV, resistant to heat inactivation; they possess the host range restrictions of RSV and are neutralized by antisera specific to the RSV subgroup. The RSV(VSV) pseudotypes are recognized as the fraction of focus-forming units which transforms chick cells resistant to infection with the strain of RSV used. Both kinds of pseudotypes are produced concomitantly with VSV synthesis. VSV(RSV) particles comprise up to 12% of the VSV progeny titer and RSV(VSV) up to 1% of the RSV titer, but pseudotype fractions varied according to the VSV mutant used for superinfection. The proportions of pseudotypes in harvests of mixed infections are not reduced by filtration through 0.2-μm pore size filters to eliminate large aggregates of virus particles, and pseudotypes are not formed by mixing pure-grown RSV and VSV particles in vitro. VSV acts as a helper virus for BH-RSV(-), which is defective in envelope antigen, but not for αBH-RSV(-), which is also defective in RNA-directed DNA polymerase activity. The titer of BH-RSV(VSV) is enhanced by the presence of the avian leukosis helper virus, RAV-1, and more than 90% of this mixed pseudotype stock is neutralized by antiserum to either VSV or RAV-1, indicating that the RSV particles bear a mosaic of both VSV and RAV-1 envelope antigens. RSV(VSV) pseudotypes transform cells of four out of five mammalian species tested. Like RSV of subgroup D and B77, the focus-forming titer of RSV(VSV) assayed on mammalian cells is 1000-fold lower than on chick cells.     

Parental G protein reincorporation by a vesicular stomatitis virus temperature-sensitive mutant of complementation group V at nonpermissive temperature.

Rescue virions obtained after superinfection of ts O45(V) Indiana serotype-infected cells by uv-irradiated vesicular stomatitis virus (VSV) at a nonpermissive temperature do not incorporate G protein synthesized at this temperature. They apparently contain G protein in their envelope since they are neutralized by homotypic antivirion and antispike sera. Rescue of ts O45(V) mutant Indiana serotype by uv-irradiated VSV New Jersey serotype is demonstrated. Ultraviolet-inactivated VSV of New Jersey serotype has therefore been used to determine the origin of the incorporated G protein molecules. The progeny-rescue virions belong genetically to VSV Indiana complementation group V. They are however neutralized by anti-New Jersey serum and not by anti-Indiana serum. Rescue ts O45(V) virions have thus probably reincorporated G protein molecules supplied by the uv-irradiated New Jersey virus. This provides further evidence suggesting that protein G is encoded for by gene V.