Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus

Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells.     

Virion functions of RNA+ temperature-sensitive mutants of Newcastle disease virus.

Virions from Newcastle disease virus mutants in four temperature-sensitive RNA+ groups were grown in embryonated hen eggs at the permissive temperature, purified, and then analyzed for biological properties at both the permissive and nonpermissive temperatures. At the permissive temperature, virions of mutants in groups B, C, and BC (11 mutants) were all lower in specific (per milligram of protein) hemagglutination, neuraminidase, and hemolysis activities compared with the wild type. These deficiencies were related to decreased amounts of hemagglutinin-neuraminidase glycoprotein in the virions. Activities of these mutant virions at both the permissive and nonpermissive temperatures were similar, indicating that hemagglutinin-neuraminidase synthesized at the permissive temperature was not temperature sensitive in function. The three group D mutants displayed a different pattern. At the permissive temperature, they had wild-type hemagglutination and neuraminidase activities but were deficient compared with the wild type in hemolysis. Again, functions were similar at both temperatures. Most of the B, C, and BC mutants had specific infectivities similar to that of the wild type despite lower hemagglutination, neuraminidase, and hemolysis functions. However, the D mutants were all less infectious. This evidence is consistent with a shared hemagglutinin-neuraminidase defect in the B, C, and BC mutants and a defect in either the F glycoprotein or the M protein in the D mutants.     

Host cell-specific growth advantage of pseudorabies virus with a deletion in the genome sequences encoding a structural glycoprotein.

Several attenuated strains of pseudorabies virus contain genomes that carry a deletion in their short unique (Us) component. The sizes of the deletions are different in the various attenuated strains; the deletions may include part of one of the inverted repeats as well as part of the Us region of the genome. In most cases, the deletion includes the gene encoding the glycoprotein gI. The attenuated strains with a deletion in their S component have a common history of having been cultivated in chicken embryo fibroblasts (CEF). We show here that passage of wild-type virus in CEF promotes the emergence of populations of virions with a deletion in their S component. The emergence of these mutants is the result of their growth advantage over the wild type and is related to the lack of expression of gI, as shown by the following. (i) The Norden strain (which has a deletion in the Us) was marker rescued to restore an intact Us. The nonrescued Norden strain had a growth advantage over the rescued Norden strain in CEF. (ii) Passage of wild-type (gI+) virus in CEF but not in rabbit kidney or pig kidney cells resulted invariably in the emergence of virions whose genomes had a deletion in the S component. (iii) Passage of a gI- mutant in CEF did not result in the emergence of such virions. The emergence of virions with a deletion in their S component thus appears to be linked to gI expression. We conclude that gI is deleterious to the growth of pseudorabies virus in CEF and that this effect is cell type specific.     

Mutation in the matrix protein of Newcastle disease virus can result in decreased fusion glycoprotein incorporation into particles and decreased infectivity.

Virus particles produced in eggs by the group D ts mutants of Newcastle disease virus at permissive temperature display low infectious and hemolytic activities (M.E. Peeples and M. A. Bratt , J. Virol. 42:440-446, 1982). These lower activities correlate with a decreased incorporation of F1+2 (fusion glycoprotein) into virus particles, compared with that for wild type. The incorporation of F1+2 into virus particles of the group D mutants is also lower than that for wild type when grown in chicken embryo cells in culture at either permissive or nonpermissive temperature. The infectivity of virions from these mutants correlates with the amounts of F1+2 in the virus particles, below a certain concentration, indicating that the quantity of F1+2 in virus particles is a determining factor in the infectivity of those particles. In addition, one of these mutants, D1, produces an M (matrix protein) which migrates at a faster rate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of four revertants of D1 have coreverted to wild-type M electrophoretic mobility, associating M with the ts lesion and the other observed phenotypes. In each of these revertants, as well as in three revertants each from D2 and D3, there has been coreversion from the low specific infectious and hemolytic activities to greater, and often wild-type, activities. There is also a coreversion for F1+2 incorporation into virions. All of the revertants incorporate F1+2 into virions more efficiently than their mutant parents. The coreversions associate those phenotypes with the ts lesion and, in the case of D1, with the M lesion as well.     

Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity.

The influenza virus hemagglutinin (HA) contains a cytoplasmic domain that consists of 10 to 11 amino acids, of which five residues have sequence identity for 10 of 13 HA subtypes. To investigate properties of these conserved residues, oligonucleotide-directed mutagenesis was performed, using an HA cDNA of influenza virus A/Udorn/72 (H3N2) to substitute the conserved cysteine residues with other residues, to delete the three C-terminal conserved residues, or to remove the entire cytoplasmic domain. The altered HAs were expressed in eukaryotic cells, and the rates of intracellular transport were examined. It was found that substitution of either conserved cysteine residue within the cytoplasmic domain did not affect the rate of intracellular transport, whereas deletion of residues within the C-terminal domain resulted in delayed cell surface expression. All the altered HAs were biologically active in hemadsorption and fusion assays. To investigate whether the wild-type HA and HAs with altered cytoplasmic tails could complement the influenza virus temperature-sensitive transport-defective HA mutant A/WSN/33 ts61S, the HA cDNAs were expressed by using a transient expression system and released virus was assayed by plaque analysis. The wild-type HA expression resulted in a release of approximately 10(3) PFU of virus per ml. Antibody neutralization of complemented virus indicated that the infectivity was due to incorporation of wild-type H3 HA into ts61S virions. Sucrose density gradient analysis of released virions showed that each of the HA cytoplasmic domain mutants was incorporated into virus particles. Virions containing HAs with substitution of the cysteine residues in the cytoplasmic domain were found to be infectious. However, no infectivity could be detected from virions containing HAs that had deletions in their cytoplasmic domains. Possible roles of the HA cytoplasmic domain in forming protein-protein interactions in virions and their involvement in the initiation of the infection process in cells are discussed.     

Simian virus 40 large T antigen host range domain functions in virion assembly.

The simian virus 40 (SV40) T antigen host range mutants dl1066 and dl1140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants dl1066 and dl1140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with dl1066 and dl1140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40% sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants dl1066 and dl1140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests that the host range blockage may result from a failure of such mutants to add VP1 to 75S assembly intermediates.     

Change of DNA near the origin of replication enhances the transforming capacity of human papovavirus BK.

A turbid-plaque-forming mutant (pm522) of human papovavirus BK, which has a small deletion at about 0.7 map unit and grows somewhat more slowly in human cells than does wild-type BK virus, transformed hamster and rat cells in culture much more efficiently than did wild-type virus. Another plaque morphology mutant, pm525, forming turbid plaques larger than those of pm522 also had a high transforming capacity. The similar difference in transforming capability between wild-type and plaque morphology viruses was observed with DNAs extracted from virions. Recombinant viruses were constructed from the wild-type DNA fragment lacking HindIII-C (0.62 to 0.73 map unit) and pm522 HindIII-C (including the origin of replication) by the molecular cloning method. Characterization of the recombinants showed that the change near the origin of DNA replication was responsible both for the altered plaque morphology and for the enhanced transforming capacity of the BK virus mutant.     

Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice.

Phenotypically complemented pseudorabies virus gp50 null mutants are able to produce plaques on noncomplementing cell lines despite the fact that progeny virions are noninfectious. To determine whether gp50 null mutants and gp50+gp63 null mutants are also able to replicate and spread in animals, mice were infected subcutaneously or intraperitoneally. Surprisingly, both gp50 mutants and gp50+gp63 double mutants proved to be lethal for mice. In comparison with the wild-type virus, gp50 mutants were still highly virulent, whereas the virulence of gp50+gp63 mutants was significantly reduced. Severe signs of neurological disorders, notably pruritus, were apparent in animals infected with the wild-type virus or a gp50 mutant but were much less pronounced in animals infected with a gp50+gp63 or gp63 mutant. Immunohistochemical examination of infected animals showed that all viruses were able to reach, and replicate in, the brain. Examination of visceral organs of intraperitoneally infected animals showed that viral antigen was predominantly present in peripheral nerves, suggesting that the viruses reached the central nervous system by means of retrograde axonal transport. Infectious virus could not be recovered from the brains and organs of animals infected with gp50 or gp50+gp63 mutants, indicating that progeny virions produced in vivo are noninfectious. Virions that lacked gp50 in their envelopes, and a phenotypically complemented pseudorabies virus gII mutant (which is unable to produce plaques in tissue culture cells), proved to be nonvirulent for mice. Together, these results show that gp50 is required for the primary infection but not for subsequent replication and viral spread in vivo. These results furthermore indicate that transsynaptic transport of the virus is independent of gp50. Since progeny virions produced by gp50 mutants are noninfectious, they are unable to spread from one animal to another. Therefore, such mutants may be used for the development of a new generation of safer (carrier) vaccines.     

Simian virus 40 maturation in cells harboring mutants deleted in the agnogene

The predominant leader region of the late 16 S mRNAs of simian virus 40 encodes a histone-like, 61-amino acid, DNA-binding protein called the agnoprotein or LP1. To test the hypothesis that this protein facilitates assembly of viral minichromosomes into virions, we have studied the synthesis of virions in cells infected with mutants deleted in this region of the SV40 genome. We found that 220 S mature virions, indistinguishable from those of wild type, were produced in cells infected with these mutants. As in wild-type-infected cells, no assembly intermediates other than 75 S chromatin were observed. However, data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced virions more slowly than cells infected with wild-type virus. Taken together with data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, these findings strongly suggest that LP1 plays a role in expediting virion assembly.     

Mutants of Sindbis virus. III. Host polypeptides present in purified HR and ts103 virus particles.

The amounts of host-encoded protein present in purified Sindbis virions of both the HR strain and of a mutant (ts103) which makes multicored particles were examined. Cells were labeled with [35S]methionine before infection and with [3H]methionine postinfection. Virions were purified by velocity sedimentation and isopycnic banding, and their polypeptides were examined by polyacrylamide gels in a sodium dodecyl sulfate-containing discontinuous buffer system. Host prelabeled material was found principally in a small number of discrete polypeptides in HR virions, which contained as little as 0.2% host-encoded protein. Virus-sized particles of mutant ts103 contained significantly more host material, and multiploid particles from ts103 infection contained up to 12% host prelabeled protein.     

Separation of Plaque-type Variants of Encephalomyocarditis Virus by Chromatography on Calcium Phosphate

Encephalomyocarditis (EMC) virus (K-2 strain) was separated into two distinct peaks of optical density by chromatography on calcium phosphate, eluting at 0.17 and 0.31 m, respectively. The virus in these peaks could not be distinguished by infectivity (plaque-forming units), hemagglutinin activity, electron microscopic appearance, sedimentation coefficient, or base ratios. However, variants producing large plaques predominated in the first peak eluted, whereas variants producing small plaques were concentrated in the second peak. When plaque-purified large and small plaque variants were chromatographed individually, each gave a single peak eluting at 0.16 and at 0.43 m, respectively. However, one small plaque type isolated was eluted in the 0.17 m region expected for large plaque variants, and not with the bulk of the small plaque variants. There were indications that EMC virus (K-2 strain) could be separated into more than two peaks by use of selected elution gradients. The possibility that there may be a relationship between plaque size and point of elution from calcium phosphate is discussed.     

Relationships Among Virus Spread, Cytopathogenicity, and Virulence as Revealed by the Noncytopathic Mutants of Newcastle Disease Virus

We have studied protein synthesis in cultured cells infected with the six noncytopathic (nc) mutants of the Australia-Victoria strain (AV-WT) of Newcastle disease virus and their plaque-forming revertants. Virus-specific polypeptides accumulated at 30 to 63% of wild-type levels in nc mutant-infected cells and between 66 and 175% of wild-type levels in revertant-infected cells. An exception was the L polypeptide, which accumulated in nc mutant-infected cells at only 5 to 20% of the levels found in wild-type infection. The reduced accumulation of the L polypeptide did not appear to be due to increased degradation of that polypeptide. A new polypeptide (X) accumulated instead of polypeptide P in cells infected with mutants nc4 or nc16 and in virions released from them. Peptide mapping identified X as an altered form of P. A revertant of mutant nc4 (nc4S1), which forms larger hemadsorbing spots, but still does not form plaques, accumulated P instead of the X polypeptide. Thus, a lesion in P can affect virus spread without affecting cytopathogenicity. Virions of mutant nc7 and two naturally occurring avirulent strains of Newcastle disease virus (NJ LaSota and B1-Hitchner) contained polypeptides (F₇ and F_A, respectively) related to, but migrating more rapidly than, F₀ in sodium dodecyl sulfate-polyacrylamide gels. As previously reported for avirulent strains, a brief treatment of nc7 virions with trypsin converted F₇ to F and increased infectivity. Similarly, culturing nc7-infected cells in the presence of trypsin facilitated fusion from within and viral spread from cell to cell. A plaque-forming revertant of nc7 still accumulated F₇ in virions, indicating that the lesions responsible for the F₇ and noncytopathic phenotypes are genetically separable. The virulent parental strain, AV-WT, exhibited a mean embryo death time of 42 h. Both the larger-spot-forming revertant of nc4 (nc4S1) and the small-plaque-forming revertant of nc7 exhibited a decrease in mean embryo death time (increase in virulence) from 74 to 63 h. A second-step, plaque-forming revertant derived from nc4S1 (nc4S1R1) exhibited a further decrease in mean embryo death time from 63 to 44 h. The results suggest that the F_A-F₇ and X lesions affect the ability of virus to spread from cell to cell. In addition, these lesions appear to be genetically separable from those responsible for the noncytopathic phenotype. However, both types of lesions cause an extension of mean embryo death time and, thus, may be relevant to virulence in vivo.     

Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.     

Comparative ultrastructural study of four reticuloendothelias viruses.

The morphology and development of four members of the reticuloendotheliosis virus group were studied by transmission electron microscopy. Virions of duck spleen necrosis virus, duck infectious anemia virus, chicken syncytial virus, and reticuloendotheliosis virus strain T are sperical with a diameter of approximately 110 nm. They are covered with surface projections about 6 nm long and 10 nm in diameter. The center-to-center distance of surface projections is about 14 nm. The budding virions contain crescent-shaped electron-dense cores 73 nm in diameter with electron-lucent centers. After release of the virions the cores apparently become condensed to 67 nm in diameter. Virions were found budding at the plasma membrane and into smooth-walled, intracytoplasmic vesicles of productively infected cells. The distribution of budding reticuloendotheliosis viruses on cells appeared random over the cell surface, and occasionally aberrant multiple forms of budding virions were observed. The virions appear to resemble mammalian leukemia and sarcoma viruses more closely than avian leukosis-sarcoma viruses.     

Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication.

Mutants of simian virus 40 (SV40) with base substitutions at or near the origin of replication of the viral genome have been constructed by bisulfite mutagenesis at the BglI restriction site of SV40 DNA, followed by transfection of cells with the BglI-resistant (BglI^r) DNA so generated. Based on plaque morphology at different temperatures, the resulting BglI^r mutants could be classified into four-groups. Class I mutants (designated ar for “altered restriction”) were indistinguishable from wild-type SV40; class II mutants (designated shp for “sharp plaque”) produced small, sharp-edged plaques; class III mutants (designated sp for “small plaque”) produced small plaques at 32 °C, 37 °C and 40 °C; and class IV mutants (designated cs for “cold sensitive”) produced small plaques at 32 °C and wild-type plaques at 37 °C and 40 °C. That the altered plaque morphology of sp and cs mutants was related to mutation at the BglI restriction site was demonstrated by co-reversion to wild-type of the plaque phenotype and BglI sensitivity. The nucleotide sequence around the original BglI site was determined in the DNA from one mutant of each class. In each case a different base-pair substitution was found, at a site outside sequences coding for SV40 proteins. When rates of replication of mutant DNAs were measured during productive infection, ar mutant DNA was synthesized at a rate comparable to that of wild-type SV40 DNA, shp mutant DNA was made at a rate exceeding that of wild-type, sp mutant DNA was synthesized at a lower rate than that of wild type. and cs mutant DNA synthesis was reduced at 32 °C, but about the same as the wild-type rate at 40 °C. These patterns of mutant DNA synthesis were unaltered in cells co-infected with mutant and wild-type virus, i.e. the defects in DNA synthesis were not trans-complementable. We conclude that the defective mutants have single base-pair changes in a cis element that determines the rate of viral DNA replication, presumably within the origin signal itself.     

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.