Complementation analysis of measles virus mutants isolated from persistently infected lymphoblastoid cell lines.

Human lymphoblastoid cell lines persistently infected with measles virus release a heterogeneous population of virions. At least 80% of the infectious particles were temperature sensitive for plaque formation at 39 degrees C. Plaque-purified temperature-sensitive mutants from four persistently infected human lymphoblastoid cell lines were shown to be heterogeneous with respect to efficiency of plating at 31 and 39 degrees C, as well as to antigen and RNA production at 39 degrees C. The heterogeneity was confirmed by complementation analysis in which 21 temperature-sensitive isolates were found to represent at least four of the five previously described complementation groups of measles virus. Two isolates complemented four reference temperature-sensitive mutants. These isolates either represent new complementation groups or are members of the fifth complementation group, group E. The majority of isolates were found to have multiple mutations, and group B mutants (RNA-) predominated. Two temperature-sensitive isolates were able to interfere with production of parental measles virus at both permissive and nonpermissive temperatures.     

Isolation and preliminary characterization of temperature-sensitive mutants of measles virus.

Twenty-four genetically stable temperature-sensitive mutants of measles virus were isolated after mutangenesis by 5-azacytidine, 5 fluorouracil, or proflavine. The restricted replication of all mutants at 39 C was blocked subsequent to cell penetration and could not be attributed to heat inactivation of virus infectivity. Complementation analysis was made possible through the use of poly-L-ornithine. The members of one complementation group exhibited wild-type RNA synthesis at the nonpermissive temperature and induced the synthesis of virus antigens. These mutants were found defective in both hemolysin antigen synthesis and cell fusion "from within," supporting the unitary hypothesis for these functions. The members of the other two complementation groups synthesized neither virion RNA nor detectable virus antigens at the nonpermissive temperature.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

Genetic and Physiological Properties of Temperature-Sensitive Mutants of Cocal Virus

Temperature-sensitive (ts) mutants of Cocal virus (VSV Cocal) were isolated after treatment with the base analogue mutagen, 5-fluorouracil. These mutants could be classified into four mutually complementing groups. Weak complementation was detected between certain pairs of VSV Cocal ts mutants and ts mutants of vesicular stomatitis virus (VSV) Indiana, but no complementation was observed with ts mutants of VSV New Jersey. Two complementing ts mutants of Chandipura virus, an unrelated rhabdovirus, did not complement any VSV mutant, Thus, ability to complement in the VSV group appears to be correlated with serological relationships.The RNA and protein-synthesizing capacities of these ts mutants have been determined, and it is possible to establish a correspondence between the VSV Cocal and the VSV Indiana complementation groups.     

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.     

Isolation and characterization of temperature-sensitive mutants of Sendai virus

Sixteen temperature-sensitive mutants of Sendai virus were isolated from mutagenized stocks (10 mutants, designated numerically) and persistently infected cultures (6 mutants, designated alphabetically). Based on complementation tests, virion-associated activities, thermal inactivation, and viral RNA and hemadsorbing antigen synthesis as well as virion production in chick lung embryo cells at nonpermissive temperature, these mutants were divided into seven groups as follows. i) HANA group mutants (ts-5, -9, -10, -201), defective in hemagglutinin-neuraminidase protein, complementation group I. ii) F group mutants (ts-18, -108), defective in hemolytic and cell-fusing activity, complementation group II. iii) Ts-43, defective in RNA polymerase activity, complementation group III. iv) Ts-23, defective in RNA polymerase activity, interfered with the other mutants in complementation tests. v) Ts-25, defective in the incorporation of hemagglutinin-neuraminidase protein into the virion at the stage of virus assembly. vi) Ts-110, belongs to F group mutants on one hand, but is considered to carry another undetermined defect. vii) C group (carrier culture-borne group) mutants (ts-a, -b, -c, -d, -e, -f), defective lesion not yet determined and belong to neither complementation group I nor II. Assignment of mutants in groups iv), v), vi), and vii) to complementation groups could not be achieved.     

The N-glycan of the SCR 2 region is essential for membrane cofactor protein (CD46) to function as a measles virus receptor.

Membrane cofactor protein (MCP) (CD46), a complement-regulatory protein, serves as a cellular receptor for measles virus. Its amino-terminal portion is composed of four short consensus repeats (SCR), three of which (SCR1, SCR2, and SCR4) carry an N-linked oligosaccharide. In order to determine the importance of the three N-glycans for the function of MCP as a measles virus receptor, we established Chinese hamster ovary (CHO) cell lines that stably express mutant MCPs lacking one of the three motifs for N glycosylation (NQ1, NQ2, and NQ4). In an additional mutant (NQ1-2), two glycosylation motifs were altered, allowing the addition of an N-linked oligosaccharide only in SCR4. The abilities of the mutant MCPs to function as measles virus receptors were analyzed with three different assays: (i) binding of measles virus hemagglutinin to MCP immobilized on nitrocellulose; (ii) binding of measles virus to CHO cells expressing wild-type or mutant MCP; and (iii) infection of the transfected CHO cells by measles virus. In all three assays, the abilities of the NQ2 and NQ1-2 mutants to serve as measles virus receptors were drastically impaired. The NQ1 and NQ4 mutants were recognized by measles virus almost as efficiently as the wild-type protein. These results indicate that the N-glycan attached to SCR2 is essential for MCP to serve as a measles virus receptor, while the oligosaccharides attached to SCR1 and SCR4 are of only minor importance.     

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.     

Complementation Analysis of Simian Virus 40 Mutants

Seventy six new temperature-sensitive mutants of simian virus 40 have been isolated. A simple modification of the standard plaquing technique permits complementation analyses to be performed readily. By using this technique the new mutants have been divided into four complementation groups.     

Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation.

Twelve temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1), representing seven complementation groups, were isolated subsequent to 5-bromodeoxyuridine mutagenesis. These mutants were identified by their inability to replicate in a line of monkey (CV-1) cells at 39 C. Seven of these mutants, representing six complementation groups, induced thymidine kinase (tk) and transformed Ltk- cells, a line of mouse L cells lacking tk, to a tk+ phenotype at both the permissive (34 C) and nonpermissive (39 C) temperatures. Thus, the defective cistrons in these six complementation groups, although necessary for lysis, have no essential function in this transformation system. Transformation by these 12 mutants was dependent on prior UV irradiation. Infection of cells with unirradiated virus under conditions which did not permit virus replication was not sufficient to allow cell transformation. Five mutants, representing two complementation groups, were tk- and were incapable of causing the tk--to-tk+ transformation at either 34 C of 39 C. The tk defects in these mutants are probably unrelated to the ts defects, since one of these complementation groups contains a tk+ member. Therefore, transformation of Ltk- cells to a tk+ phenotype by HSV-1 requires an active viral tk gene. One complementation group was represented by a single tk- member. The role of this cistron in transformation remains undetermined since the primary block to transformation is presumed to be the tk- phenotype. Mutants representing the seven complementation groups were unable to replicate at 39 C in two lines of HSV-1-transformed cells, indicating that the activities of resident wild-type copies of the defective cistrons, if present, could not be detected by complementation.     

Screening procedure for complementation-dependent mutants of vesicular stomatitis virus.

To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.     

Antigenic determinants of measles virus hemagglutinin associated with neurovirulence.

The biological activity of monoclonal antibodies specific for the hemagglutinin protein of measles virus strain CAM recognizing six epitope groups according to their binding properties to measles virus strain CAM/R401 was investigated in vivo in our rat model of measles encephalitis. When injected intraperitoneally into measles virus-infected suckling rats, some monoclonal antibodies modified the disease process and prevented the necrotizing encephalopathy seen in untreated animals. The analysis of measles virus brain isolates revealed emergence of variants that resisted neutralization with the passively transferred selecting monoclonal antibody but not with other monoclonal antibodies. Monoclonal antibody escape mutants were also isolated in vitro, and their neurovirulence varied in the animal model. Sequence data from the hemagglutinin gene of measles virus localize a major antigenic surface determinant of the hemagglutinin protein between amino acid residues 368 and 396, which may be functionally important for neurovirulence. The data indicate that the interaction of antibodies with the measles virus H protein plays an important role in the selection of neurovirulent variants. These variants have biological properties different from those of the parent CAM virus.     

Genetic complementation of propionyl-CoA carboxylase deficiency in cultured human fibroblasts.

Propionyl-CoA carboxylase (PCC) deficiency is an inherited metabolic disorder showing considerable variability of expression. We have investigated the possibility that there is a genetic basis for the clinical heterogeneity in this disorder by examining complementation in Sendai virus mediated heterokaryons of mutant fibroblast strains. Restoration of PCC activity was monitored in individual multinucleate cells in situ using a radioautographic procedure which detects the incorporation of 14C-propionate into trichloracetic acid precipitable material. Each mutant strain incorporated negligible amounts of radioactivity compared to control strains. Activity was not restored when different mutants were mixed without virus or when homokaryons were produced by self-fusion. Seven mutant strains were fused in all pairwise combinations and examined for increased 14C-propionate incorporation in heterokaryons. Two main complementation groups were revealed. One group was composed of three mutants. The other was a complex group composed of four mutants in which intragroup complementation was demonstrated. Two mutants showing excellent complementation by radioautography were examined for complementation by the direct assay of PCC ACTIVITY. The enzyme activity of virus-treated preparations with 23% multinucleate cells was 183 U (pmol/min/mg protein) compared to 16 U for the untreated mixture (normal range 450-850 u). We conclude that PCC deficiency resulted from mutations of heterogeneous origin, although the classification of mutants into complementation groups did not correlate with patterns of clinical heterogeneity.     

Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2.

Twenty-three complementation groups of herpes simplex virus type 1 (HSV-1) and 20 of HSV-2 were identified by qualitative and quantitative complementation analysis from among 43 temperature-sensitive (ts) mutants of HSV-1 and 29 ts mutants of HSV-2 which had been isolated independently in 10 laboratories.     

DNA-negative temperature-sensitive mutants of herpes simplex virus type 1: patterns of viral DNA synthesis after temperature shift-up.

Temperature-sensitive mutants of herpes simplex virus type 1 belonging to four DNA- complementation groups exhibited two distinct patterns of viral DNA synthesis after shift-up to the nonpermissive temperature. In cultures infected with mutants belonging to complementation groups A, C, and D, little or no viral DNA was synthesized after shift-up. In cultures infected with a mutant in complementation group B, nearly normal amounts of viral DNA were synthesized after shift-up.     

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes.     

BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus.

The process by which Sindbis virus excludes superinfecting homologous virus was investigated with the use of temperature-sensitive mutants. Mutants in two RNA-negative complementation groups were found to be defective in their ability to establish interference at the nonpermissive temperature. These mutants were unable to establish interference in a mixed infection (complementation), suggesting that both were defective in a common gene product. Homologous interference was found to block the replication of superinfecting virus after attachment, penetration, and translation of the nonstructural genes encoded in the virus RNA. The production of nonstructural gene products of superinfecting wild-type virus was found to enhance the replication of certain RNA- temperature-sensitive interfering viruses at the permissive and the nonpermissive temperature. The ability of certain RNA- mutants to establish homologous interference and to demonstrate enhanced growth after superinfection with wild-type virus was interpreted to produce a model implicating both virus and host components in the establishment of homologous interference and in the replication of Sindbis virus RNA.     

Isolation and Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus, New Jersey Serotype

Forty-eight temperature-sensitive (ts) mutants have been isolated from a wild-type strain of the New Jersey serotype of vesicular stomatitis virus (VSV) after exposure to the base analogue mutagen 5-fluorouracil. Of these mutants, 47 have been classified into 6 nonoverlapping complementation groups containing 21, 17, 4, 3, 2, and 1 mutant, respectively (1 mutant remaining unallocated). The ribonucleic acid (RNA) phenotype of 23 of these mutants has been established. Four of the six groups contain one or more mutants unable to synthesize detectable amounts of viral RNA under restrictive conditions (39 C). No complementation was observed in mixed infection with ts mutants from the five established complementation groups of the Indiana serotype of VSV.     

Genetic analysis of murine hepatitis virus strain JHM.

We performed a genetic analysis of 37 temperature-sensitive mutants of murine hepatitis virus strain JHM. Of our mutants, 32 did not induce murine hepatitis virus-specific RNA synthesis in infected cells at the restrictive temperature, 39 degrees C. By complementation testing we have identified at least seven nonoverlapping complementation groups. Six of the genes identified in this way are required for murine hepatitis virus-specific RNA synthesis. The seventh complementation group is made up of five mutants which induced virus-specific RNA synthesis at 39 degrees C.     

Isolation and preliminary characterization of temperature-sensitive mutants of Newcastle disease virus.

Temperature-sensitive (ts) mutants of Newcastle disease virus have been isolated and characterized genetically (complementation), biochemically (RNA synthesis) and biologically (fusion from within and hemadsorption). Fifteen of these mutants have been divided into five complementation groups. Groups A (five mutants) and E (one mutant) are ts for RNA synthesis (RNA-) as well as for the other functions. Group B contains four RNA+ mutants of which one is ts for fusion, one for hemadsorption and two for neither function. Group C contains one RNA+ mutant which is a poor cell fuser. Group D contains two RNA+ mutants which are ts for fusion. In addition, two noncomplementing mutants (group BC) fail to complement both group B and group C mutants while exhibiting complementation with mutants in groups A, D, and E.