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... Efficient packaging of defective interfering particles by viral capsid proteins supplied by nondefective homologous viruses during coinfection demonstrated that encapsidation could occur in trans. Earlier work showed that poliovirus and other enterovirus capsid proteins could trans-encapsidate each other's RNAs when cells were coinfected with the two different viruses (15,33). It has also been shown that poliovirus capsid protein expressed from a recombinant vaccinia virus could provide capsid protein in trans (4). ...
A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 x 10(6) infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.
... Therefore, we had to publish our study with some delay in Russian (Agol and Shirman, 1965). Ironically, both our competitors similarly divided their analogous results between two papers, and their publications preceded ours (Holland and Cords, 1964;Wecker and Lederhilger, 1964b). As a partial consolation, our Russian paper was selected by a US journal for translation into English (Agol and Shirman, 1966). ...
This Invited Review is a kind of scientific autobiography based on the presentation at the Symposium "Viruses: Discovering Big in Small" held in honor of the author's 90th birthday (Moscow, March 2019).
... Injured RNA genomes, even if they are dead, as such, may survive for at least some generations due to complementation, i.e., help provided in trans by proteins (or RNA cis-elements) encoded by their coinfecting viruses. This mechanism of cooperative interaction was described long ago for the case of drug-sensitive and drug-resistant (or -dependent) picornaviruses (293)(294)(295)(296)(297), but its wider biological relevance became especially appreciated after the realization that viral populations are represented by quasispecies, i.e., swarms of closely related but distinct individuals (10,14,32,73,(298)(299)(300)(301). The prolonged survival of impaired genomes owing to intrapopulation complementation may provide time for their adaptive remodeling, resulting in the restoration of their capacity for independent existence. ...
Reproduction of RNA viruses is typically error-prone due to the infidelity of their replicative machinery and the usual lack of proofreading mechanisms. The error rates may be close to those that kill the virus. Consequently, populations of RNA viruses are represented by heterogeneous sets of genomes with various levels of fitness. This is especially consequential when viruses encounter various bottlenecks and new infections are initiated by a single or few deviating genomes. Nevertheless, RNA viruses are able to maintain their identity by conservation of major functional elements. This conservatism stems from genetic robustness or mutational tolerance, which is largely due to the functional degeneracy of many protein and RNA elements as well as to negative selection. Another relevant mechanism is the capacity to restore fitness after genetic damages, also based on replicative infidelity. Conversely, error-prone replication is a major tool that ensures viral evolvability. The potential for changes in debilitated genomes is much higher in small populations, because in the absence of stronger competitors low-fit genomes have a choice of various trajectories to wander along fitness landscapes. Thus, low-fit populations are inherently unstable, and it may be said that to run ahead it is useful to stumble. In this report, focusing on picornaviruses and also considering data from other RNA viruses, we review the biological relevance and mechanisms of various alterations of viral RNA genomes as well as pathways and mechanisms of rehabilitation after loss of fitness. The relationships among mutational robustness, resilience, and evolvability of viral RNA genomes are discussed.
An early stage of encephalomyocarditis (EMC) virus multiplication, probably the effective release of viral genome, is impaired in the majority of cells of a HeLa subline. Poliovirus, replicating or not, induces an actinomycin-sensitive conversion of the nonpermissive cells into permissive ones. These conclusions are based on the following observations. At moderate multiplicities of infection with EMC virus, only a small fraction of the cells in the population undergoes productive infection, as revealed by either infectious centers or the immunofluorescent techniques. The restriction in the majority of the cells can be overcome either by an increase in the multiplicity of infection or by coinfection with poliovirus. Replication of the helper virus RNA does not seem to be a prerequisite for the stimulation of EMC virus multiplication, since the enhancing effect can be obtained with guanidine-sensitive strains of poliovirus in the presence of guanidine or with guanidine-dependent strains in the absence of the drug. On the other hand, simultaneous coinfection with poliovirus fails to stimulate EMC virus reproduction in the presence of aurantine, a preparation of actinomycins. If, however, the cells are preinfected with the helper virus under conditions preventing its replication, the subsequent superinfection with EMC virus proceeds efficiently even in the presence of the antibiotic. Preinfection with poliovirus does not affect appreciably either adsorption and eclipse of EMC virus or the infectious center formation by EMC virus RNA.
Poliovirus RNA replication is known to be inhibited by millimolar concentrations of guanidine. A variety of guanidine-resistant (gr) and guanidine-dependent (gd) poliovirus strains were selected, and mutations responsible for the phenotypic alterations were mapped to distinct loci of the viral NTP-binding pattern containing protein 2C. Together with already published results, our data have demonstrated that the overwhelming majority of guanidine mutants of poliovirus 2C can be assigned to one of the two classes, N (with a change in Asn179) or M (with a change in Met187). As inferred from the structure/function relations in other NTP-binding proteins, both these “main” mutations should reside in a loop adjoining the so-called B motif known to interact with the Mg2+, involved in the NTP splitting. In classes M (always) and N (not infrequently), these B motif mutations were combined with mutations in, or close to, motif A (involved in binding of the NTP phosphate moieties) and/or motif C (another conserved element of a subset of NTP-binding proteins). These data strongly support the notion that the region of polypeptide 2C involved in the NTP utilization is affected by the guanidine mutations and by the presence of the drug itself. The mutations, however, never altered highly conserved amino acid residues assumed to be essential for the NTP binding or splitting. These facts and some other considerations led us to propose that guanidine affects coupling between the NTP binding and/or splitting, on the one hand, and the 2C function (related to conformational changes), on the other. Both N and M classes of mutants contain gr and gd variants, and the gr/gd interconversion as well as modulations of the guanidine phenotype can be caused by additional mutations within each class; sometimes, these additional substitutions are located far away from the “main” mutations. It is suggested that the target for guanidine action involves long-range tertiary interactions.
The replication of Mouse Elberfeld (ME) virus was accelerated when HEp-2 cells were mixedly infected with poliovirus in the presence of guanidine. The latent period of the replication of ME virus was shortened by 3 h when cells were preinfected for at least 2 h with poliovirus and inhibited by guanidine. Simultaneous infection with poliovirus and ME virus resulted in a shortening by 1 h of the latent period of ME virus replication. The accelerated replication of ME virus was shown to be due to modification and exploitation of a membrane complex induced by poliovirus in the presence of guanidine; on superinfection ME virus successively modified this poliovirus-induced complex of 470S ("light' complex) into a "heavy' complex of 700S specific for ME virus.
The biochemical basis for variation in foot-and-mouth disease virus (FMDV) has been explored by analysis of the virus RNA and the virus-induced and structural proteins of three isolates of the virus. Two of the isolates were from serotype A and the third was from serotype O. Hybridization studies of the RNAs showed greater than 80% homology between the two type A viruses and about 65% homology between the two type A viruses and the virus of type O. The ribonuclease T1 maps of the three viruses gave distinct patterns typical of FMDV, but did not show that any two of the three viruses were more closely related. The virus-induced primary translation products, P88, P52 and P100 isolated from infected cells, were compared by tryptic peptide analysis. Combinations of 3H- and 14C-leucine-labelled polypeptides were hydrolysed with trypsin and resolved on an ion-exchange column. Much greater differences were found in P88 than in P52 or P100, indicating that the major variation occurs in the region of the genome coding for the structural proteins. Similar analysis of combinations of the structural proteins of the three viruses showed that there were differences in VP1, VP2 and VP3 and these results were supported by those obtained by PAGE analysis of the Staphylococcus aureus V8 protease cleavage products.
Four different temperature-sensitive (ts) mutants derived from the Mahoney strain of poliovirus type 1, were crossed in an infectious center recombination test. Evidence for recombination was obtained in three crosses, with a different segregation of an unselected marker, resistance to guanidine, in each case. Evidence for genetic complementation between ts mutants was not found, except with one set of RNA- mutants, ts 221 and ts 035. The marked virus yield enhancement which was observed in cells mixedly infected by these two mutants resulted from a nonreciprocal rescue of ts 035 by ts 221. The effects of ts 221 input multiplicity and of guanidine inhibition of viral RNA replication on the rescue were analyzed. The results showed that yield enhancement of ts 035 in mixed infection could be correlated to the low level RNA replication of ts 221 at the nonpermissive temperature.
Foot-and-mouth disease virus (FMDV) and bovine enterovirus (BEV) were used for simultaneous dual infection of calf kidney tissue cultures. In the harvest fluid a viral agent was found that had the FMDV genome, but (1) was neutralized by BEV-antiserum and not by FMDV-antiserum; (2) had the acid-stability of BEV and not FMDV; (3) had the buoyant density of BEV (1.35 class) and not FMDV (1.45 class); and (4) had the s-rate class (140–150 S) of both parental viruses. Such a particle meets criteria for simple genomic masking. Its ultracentrifugal properties show that the protein coat influenced the buoyant density in CsCl more than nucleic acid, presumably because of permeability to salt.
Evidence is presented that poliovirus particles with a single lethal hit by hydroxylamine do not induce in host cells either inhibition of cellular protein synthesis or viral ribonucleic acid (RNA) replication. The RNA of these viruses is not replicated even if the cells are simultaneously infected with both active and inactivated viruses. The damaged viral RNA seems to have lost both its template function and its function in the translation of normal viral proteins.
Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.
Guanidine (1.5mM) inhibits viral RNA synthesis in cells that are actively producing virus. It was found that synthesis of viral RNA becomes progressively less inhibitable by guanidine after the fourth hour in the viral growth cycle when the rate of viral RNA synthesis diminishes.Guanidine added at 3 hours inhibited the synthesis of single-stranded RNA faster than synthesis of double-stranded RNA; however, 35 S single-stranded RNA was still the major RNA moiety formed. While there was a net reduction in the cumulative radioactivity of ribonuclease-resistant RNA in the replicative intermediate, there was an increase in the amount of radioactivity in the double-stranded replicative form after guanidine was added.These results indicate that guanidine does not inhibit growth and completion of viral RNA chains, but blocks the initiation of new chains.
Crowell, Richard L. (Hahnemann Medical College, Philadelphia, Pa.). Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference. J. Bacteriol.
198–204. 1966.—Exposure of HeLa cells to high levels of coxsackievirus B3 produced cells which were refractory to attachment of coxsackievirus B1, whereas poliovirus T2 attached normally. Under similar conditions, poliovirus T2 was found to interfere with the attachment of poliovirus T1 to HeLa cells without affecting the attachment rate of coxsackievirus B3. The data confirm earlier findings that the receptor sites on HeLa cells, which bind members of group B coxsackieviruses, are distinct from those for polioviruses. Quantitatively, coxsackieviruses B1 and B3 were found to be mutually exclusive in the attachment interference assay to suggest that they compete for the same receptors on the HeLa cell surface. The finding that input multiplicities of B3 virus which exceeded 500 saturated the homologous viral receptors of HeLa cells was unexpected, but was consistent with the results of interference assays. Excessive amounts of input virus did not, however, inhibit eclipse of homologous cell-associated virus. Attachment interference between enteroviruses occurred even though the interfering virus was eclipsed prior to addition of challenge virus. The finding that enterovirus attachment interference was reversible with acid pH suggested that attachment and eclipse of enterovirus does not result in a permanent alteration of the cell membrane and that these events occur at the cell surface.
The cell-free synthesis of virus specific RNA by foot-and-mouth disease virus (FMDV) RNA polymerase was inhibited by antisera from infected guinea pigs and cattle, by γ-globulin fractions from sera of infected guinea pigs, and by antibody isolated from antibody-antigen precipitates. Deoxycholate, at a concentration of 0.5%, increased the inhibitory effect from 20% to 90%. Ribonucleic acid profiles of antibody-inhibited enzyme products showed a small peak corresponding to 20 S RNase-resistant material and no 37 S viral RNA peak as usually seen in the in vitro deoxycholate pattern.Antisera from animals vaccinated with inactivated virus, which differed from that obtained by infection in not possessing antibody to a third antigenic component, did not inhibit polymerase activity. This third antigenic component was previously found to be a nonviral constituent associated with infection. Preparations of antisera from infected animals from which antibody to this antigen had been removed in stepwise amounts, showed corresponding decreases in inhibition of polymerase activity. Polymerases from FMDV types A and O appeared to be immunologically related, whereas that from type SAT 2 did not.The evidence indicates that FMDV-RNA polymerase may be the third antigenic component.
Foot-and-mouth disease is an economically devastating disease of livestock caused by foot-and-mouth disease virus (FMDV). Vaccination is the most effective control measure in place to limit the spread of the disease; however, the success of vaccination campaigns is hampered by the antigenic diversity of FMDV and the rapid rate at which new strains emerge that escape pre-existing immunity. FMDV has seven distinct serotypes, and within each serotype are multiple strains that often induce little cross-protective immunity. The diversity of FMDV is a consequence of the high error rate of the RNA-dependent RNA polymerase, accompanied by extensive recombination between genomes during co-infection. Since multiple serotypes and strains co-circulate in regions where FMDV is endemic, co-infection is common, providing the conditions for recombination, and also for other events such as trans-encapsidation in which the genome of one virus is packaged into the capsid of the co-infecting virus. Here, we demonstrate that the co-infection of cells with two FMDVs of different serotypes results in trans-encapsidation of both viral genomes. Crucially, this facilitates the infection of new cells in the presence of neutralizing antibodies that recognize the capsid that is encoded by the packaged genome.
We constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone. The derived viral strains carrying the mutations exhibited a variety of distinct plaque phenotypes. Thus, we were able to examine genetic complementation between different pairs of mutants by comparing the yields of progeny virus in mixed and single infections. Two mutants bearing lesions in the 2A and 3A regions of the genome, which are defective in the inhibition of host cell translation and the synthesis of viral RNA, respectively, could be rescued efficiently by genetic complementation; three replication-deficient mutants containing insertions in the 2B, 3D (replicase), and 3'-untranslated regions could not. Both the 2A and 3A mutants could be rescued by each other and by all of the other mutants tested. Because yield enhancement was apparent well before the completion of a single infectious cycle, it is likely that complementation of both mutants involved early diffusion of functional products. These data provide the first unambiguous evidence that the nonstructural portion of the poliovirus genome contains multiple complementation groups. The data also suggest that certain nonstructural functions act only in cis.
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.
Phenotypic mixing as a distinct phenomenon was first recognized in bacteriophages. For some time it was considered to be of marginal importance. However, it was gradually recognized that phenotypic mixing is very common in dual infections of cells with two viruses belonging to one group, or even to different groups. Most of the published work has been done with animal viruses, but the phenomenon is known in plant viruses too. Phenotypic mixing is of major importance in oncornaviruses where it enables the rescue of defective viral genomes and the detection of only partially expressed viruses. It now appears that phenotypic mixing occurs in some instances also between members of different viral groups. It was shown that some plaque forming viruses may acquire surface antigens of viruses that otherwise are difficult to detect. The subject of viral pseudotype formation and phenotypic mixing is reviewed.
Deep hypersaline anoxic basins in the Mediterranean Sea are a legacy of dissolution of ancient subterranean salt deposits
from the Miocene period. Our study revealed that these hypersaline basins are not biogeochemical dead ends, but support in
situ sulfate reduction, methanogenesis, and heterotrophic activity. A wide diversity of prokaryotes was observed, including
a new, abundant, deeply branching order within the Euryarchaeota. Furthermore, we demonstrated the presence of a unique, metabolically active microbial community in the Discovery basin,
which is one of the most extreme terrestrial saline environments known, as it is almost saturated with MgCl2 (5 M).
The RNA-dependent RNA polymerase induced in BHK 21 cells by infection with foot-and-mouth disease virus has been isolated from the replication complex. It contains a major, virus-coded protein with mol. wt. 56 000 which appears from serological studies and tryptic peptide mapping to be the same as the virus infection associated (VIA) antigen and the protein P56 found in cells infected with the virus. Other virus coded proteins and a host cell protein were present in the partially purified replication complex but were removed by digestion with ribonuclease T1, leaving only the major virus coded protein. The tryptic peptide maps of the VIA antigen of the seven serotypes of the virus were similar, suggesting a high level of conservation in that region of the genome coding for the RNA polymerase of each type.
Gemischte Infektion von primren Klbernieren-Kulturen mit temperatursensitivem (ts) und temperaturresistentem (tr) Maul- und Klauenseuche-Virus fhrt bei 40C-Bebrtung zu Rescue des bei 40 C nicht vermehrungsfhigen ts-Virus. Das gleiche Phnomen tritt bei gemischter Infektion von Suglingsmusen auf, denen gleichzeitig avirulentes ts-Virus und virulentes tr-Virus injiziert wird. Dabei kann es zur Vermehrungshemmung des tr-Virus kommen (Interferenz). Rescue ist innerhalb der geprften Typen O, A und C nicht typenspezifisch. Neutralisationsversuche mit typenspezifischen Immunseren ergaben, da Rescue-Virus nur Hllprotein des vermehrungsfhigen Virusstammes enthlt. Genommaskiertes Virus verhlt sich bei der Thermoinaktivierung wie der Helfer-Virusstamm, von dem die Proteinhlle stammt.A mixed infection of primary calf kidney cultures with temperature sensitive (ts) and temperature resistant (tr) foot-and-mouth, disease virus and subsequent incubation at 40 C results in a rescue of the virus, which, otherwise does not replicate at 40 C. The same rescue phenomenon occurs in suckling mice, which are simultaneously injected with avirulent ts-virus and virulent tr-virus. This may even lead to an inhibition of tr-virus (interference). The rescue phenomenon is not type specific within the investigated types O, A and C. Neutralization tests with type specific immune sera showed that the rescued virus contains capsid protein of the tr-virus only. When thermal inactivation is applied, the virus with a masked genome has the same behaviour as the helper virus strain, which provides the capsid protein.
Picornaviruses have long seemed to have many genes. The size of the genome compared with the “average” gene product suggested about ten for poliovirus (Fenner, 1968), which causes at least a dozen synthetic and degenerative changes in the host cell. Many viral polypeptides are found, originally about 14 (Summers et
al.,1965) and now more than 34 (Abraham and Cooper, 1975a). Other picornaviruses are very similar.
The term picornavirus, i.e., small RNA virus, was proposed in 1963 (International Enterovirus Study Group, 1963) as a category encompassing the small, ether-resistant, polyhedral, RNA-containing viruses of man and animals. Having survived for more than a decade in the face of numerous new proposals for the classification of viruses (Lwoff and Tournier, 1971), the term appears to have become firmly rooted in the vocabulary of virology.
Viral RNA synthesis was assayed in HeLa cells transfected with nonviable poliovirus RNA mutated in the genome-linked protein VPg-coding region. The transfecting RNA was transcribed in vitro from full-length poliovirus type 1 (Mahoney) cDNA containing a VPg mutagenesis cartridge. Hybridization experiments using ribonucleotide probes specific for the 3' end of positive- and negative-sense poliovirus RNA indicated that all mutant RNAs encoding a linking tyrosine in position 3 or 4 of VPg were replicated even though no virus was produced. VPg, but no VPg precursor, was found to be linked to the 5' end of the newly synthesized RNA. Encapsidated mutant RNAs were not found in transfected-cell lysates. After extended maintenance of transfected HeLa cells, a viable revertant of one of the nonviable RNAs was recovered; the revertant lost the lethal lesion in VPg by restoring the wild-type amino acid, but it retained all other nucleotide changes introduced during construction of the mutagenesis cartridge. Mutant RNA encoding phenylalanine or serine rather than tyrosine, the linking amino acid in VPg, was not replicated in transfected cells. A chimeric mutant containing the VPg-coding region of coxsackievirus within the poliovirus genome was viable but displayed impaired multiplication. A poliovirus-coxsackievirus chimera lacking a linking tyrosine in VPg was nonviable and replication-negative. The results indicate that a linkage-competent VPg is necessary for poliovirus RNA synthesis to occur but that a step in poliovirus replication other than initiation of RNA synthesis can be interrupted by lethal mutations in VPg.
1.1. Initial velocities for the reaction catalyzed by DPNH-cytochrome c reductase from pig heart sarcosomes have been determined at 14° as a function of DPNH, cytochrome c and hydrogen ion concentration.2.2. Maximum velocities and Michaelis constants have been calculated from the data over a pH range of 7 to 126.96.36.199. At constant pH, the kinetic results follow the rate equation which indicates that the enzymic reaction may be treated as a “two substrate” case even though the overall stoichiometry of the reaction requires three substrate molecules.4.4. The pH-dependence of the maximum velovity indicates that three ionizing groups are involved in the enzymic reaction. Furthermore, the pH-dependence of the maximum velocity-Michaelis constant ratio shows that two of the three groups are in the enzymically active site and are associated with the oxidation of DPNH. The third ionizing group is involved in the reduction of cytochrome c. It is impossible to determine kinetically whether this third grouo is associated with the enzyme or with the cytochrome itself.5.5. Ionization constants for the groups involved and the so-called pH-indepedent kinetic parameters have been calculated.6.6. The β-deuterium labelled reduced DPN has been made by the stereospecific exchange reaction catalyzed by the enzyme. Use of this material as a substrate shows that the Michaelis constant for DPNH in unaffected but that the maximum velocity is decreased approximately 2.3 fold.
Low concentrations of p-fluorophenylalanine (5–10 μg/ml) inhibit the maturation of Western equine encephalomyelitis and poliomyelitis viruses. Much higher concentrations are required (125 μg/ml) to inhibit the synthesis of the respective infectious RNA. By means of fluorescent antibodies against poliovirus, it was found that the formation of the viral antigens and the viral RNA are inhibited in a parallel manner by FPA. The implications of these findings suggesting a mutual dependency of viral protein and viral RNA synthesis are discussed.
The bacteriophage P22 has a short tail with a hexagonal base plate but no contractile sheath. It is not adsorbed on a mutant strain () of its host Salmonella typhimurium (St). A mutant P22h, forming faint plaques on and clear plaques on St, is indistinguishable morphologically from P22. Preparations of P22 also contain a small proportion of a long-tailed phage, P221, which forms plaques on but not on St, by which it is not adsorbed. Different strains of P22, containing markers like c+, c1 and c2 that affect lysogenization, produce P221 strains with corresponding markers. Neither P22 nor P22h cross reacts serologically with P221. However, mixed infection of by P22h and P221 produces masked genomes: particles carrying P22h genomes in P221 capsids and P221 genomes in P22h capsids. Moreover, P22h markers can be transferred to P221 genomes and vice versa. The existence of these stable hybrids may indicate that P221 represents a morphologically and serologically distinct mutant of P22. Alternatively, there may be present in St a defective prophage whose defects can be rectified by recombination with P22 to yield P221.
Interference is induced in HeLa cells by active poliovirus type 1 against superinfection by active type 2, and vice versa. The resistance is such that even high challenge doses are excluded from initiating growth. Challenge virus labeled with P32 adsorbs to resistant cells, and the adsorbed virus is extensively degraded. The rate and extent of breakdown are similar to that which occurs with virus infecting normal cells. The interference blocks the development of detectable amounts of challenge viral antigens, infective RNA and protein coat material.Interference against high challenge doses is induced after interfering virus enters the cell (as measured by the loss of antiserum sensitivity of infective centers) and very shortly before infective RNA is formed. The interval between induction of complete resistance and onset of detectable RNA formation is about 30–40 minutes, irrespective of the size of the interfering dose. However, the time lag between adsorption and induction of interference is inversely related to the size of the interfering dose. A single infective particle of interfering virus induces interference.Interference against low challenge doses is established somewhat earlier than against high doses. A normal mixed yield of the first and the superinfecting viruses is produced in single cells which resist low challenge doses but not high doses.