Intrinsic Interference: a New Type of Viral Interference

The hemadsorption-negative plaque test has revealed a new type of viral interference, termed intrinsic interference. Several unrelated types of noncytopathic viruses were shown to induce in infected host cells a state of interference unique in being directed solely against superinfection by Newcastle disease virus (NDV). The NDV-refractory state arises only in those individual cells of a population actually infected by the inducing virus, and presumably results from the action of a protein(s) coded for by the viral genome. Thus, intrinsic interference differs fundamentally from that mediated by an extrinsic protein detectable under conditions favoring resistance to a broad spectrum of viruses and characteristic of interference induced by interferon, the latter being coded for by the cell genome. Intrinsic interference is defined as a viral genome-induced cellular state of resistance to challenge by high multiplicities of NDV, coexistent with a state of susceptibility to a broad spectrum of other viruses, similarly tested at high multiplicities. The capacity to induce intrinsic interference was demonstrated with rubella virus, Sindbis virus (arbovirus, group A), West Nile virus (arbovirus, group B), poliovirus (MEF, type 2), the lactic dehydrogenase virus (Riley's agent), and an unidentified nonhemadsorbing, noncytopathic adventitious virus. A state of intrinsic interference was also observed in the V5 line of mouse cells carrying a murine leukemia virus, probably resulting from some heretofore unsuspected contaminating virus. The molecular basis for intrinsic interference is not known, but it appears to involve a step in the NDV growth cycle beyond that of viral attachment, entry, and eclipse.     

Defective Influenza Viral Ribonucleoproteins Cause Interference

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.     

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.     

Molecular cloning of Mus dunni endogenous virus: an unusual retrovirus in a new murine viral interference group with a wide host range.

Mus dunni endogenous virus (MDEV) is activated from cells of the Asian wild mouse M. dunni (also known as Mus terricolor) in response to treatment with either 5-iodo-2'-deoxyuridine or hydrocortisone. MDEV represents a new murine retrovirus interference group and thus appears to use a different receptor for entry into cells than do other murine retroviruses. Here we show that MDEV is also not in the gibbon ape leukemia virus or RD114 virus interference groups. A retroviral vector with an MDEV pseudotype was capable of efficiently infecting a wide variety of cells from different species, indicating that the MDEV receptor is widely expressed. We isolated a molecular clone of this virus which exhibited no hybridization to any cloned retrovirus examined, suggesting that MDEV has an unusual genome. One copy of a possible retrovirus element that weakly hybridized with MDEV was present in the genomes of laboratory strains of mice, while no such elements were present in other species examined. A virus activated by 5-iodo-2'-deoxyuridine from cells of a BALB/c mouse, however, was not related to MDEV by either hybridization or interference analyses.     

Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference

Crowell, Richard L. (Hahnemann Medical College, Philadelphia, Pa.). Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference. J. Bacteriol. 91:198-204. 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.     

应用RNAi技术培育抗2种病毒病的转基因烟草

分别提取烟草普通花叶病(TMV)和烟草黄瓜花叶病（CMV）的病毒RNA。经反转录和外壳蛋白阅读框PCR扩增,获得TMV和CMV外壳蛋白基因cDNA, 分别进行两种病毒已知株系cDNA序列比对获得各自的保守序列,设计干涉序列,将干涉片段扩增产物连接到pMD18-T的相邻酶切位点,制备融合序列,并将其正向和反向序列插入pUCCRNAi载体,再转化到pCAMBIA2300-35S-OCS表达载体中。利用农杆菌LBA4404侵染烟草K326,获得3份含有TMV和CMV外壳蛋白基因干涉序列的转化材料,经分子鉴定证实干涉序列已导入烟草,并采用荧光定量PCR技术对其mRNA表达差异进行分析。抗病性调查表明转化烟株对TMV和CMV抗性都显著增强。     

Homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units.

Lymphocytic choriomeninigitis (LCM) virus defective interfering (DI) particles form foci of protected cells in a monolayer under an agarose-containing overlay medium. Foci originate from one cell dually infected with at least 1 interference focus-forming unit and infectious virus. As a result, an interfering factor is produced and released which interacts with neighboring cells, thereby protecting them against cytopathic lysis by challenge virus. The property of individual LCM virus DI particles to induce countable foci has been made the basis of quantitative assay that is comparable in every respect to the plaque assay of infectious virus and is much more sensitive and probably more accurate than other procedures used to measure LCM virus DI particles. LCM virus was passaged, undiluted, 10 times in cell cultures. When yields were analyzed as to concentrations of PFU and interference focus-forming units, both entities were found to fluctuate with the pattern expected from theoretical considerations.     

Protection against retroviral diseases after vaccination is conferred by interference to superinfection with attenuated murine leukemia viruses.

Cell cultures expressing a retroviral envelope are relatively resistant to superinfection by retroviruses which bear envelopes using the same receptor. We tested whether this phenomenon, known as interference to superinfection, might confer protection against retroviral diseases. Newborn mice first inoculated with the attenuated strain B3 of Friend murine leukemia virus (F-MuLV) were protected against severe early hemolytic anemia and nonacute anemiant erythroleukemia induced by the virulent strain 57 of F-MuLV. Vaccinated animals were also protected as adults against acute polycythemic erythroleukemia induced upon inoculation with the viral complex containing the defective spleen focus-forming virus and F-MuLV 57 as helper virus. Animals were inoculated as newborns, which is known to induce immune tolerance in mice, and the rapid kinetics of protection, incompatible with the delay necessary for the immune response to develop, indicated that protection was not due to an immune mechanism but rather was due to the rapid and long-lasting phenomenon of interference. This result was confirmed by combining parental and envelope chimeric MuLV from different interference groups as vaccinal and challenge viruses. Although efficient protection could be provided by vaccination by interference, we observed that attenuated replication-competent retroviruses from heterologous interference groups might exert deleterious synergistic effects.     

Interference is controlled by segment 2 and possibly by segment 8 of the nondefective interfering influenza virus variant A/FM/1/47-MA.

On mouse adaption of A/FM/1/47, a variant, A/FM/1/47-MA (FM-MA), that had acquired the properties of increased virulence and interference was produced. Coinfection of cells with FM-MA and prototype strains of influenza virus yielded > 100-fold more FM-MA virus than prototype virus, whereas coinfection with the same prototype strains and the parental A/FM/1/47 virus produced equivalent yields, indicating that FM-MA had acquired mutations that confer the property of interference during mouse adaption. FM-MA is a nondefective interfering virus that grows to a high titer in vivo and in vitro. It has previously been shown that segments 4, 7, and 8 and possibly segment 5 account for the increased virulence. In this study we show by genetic analysis of FM-MA x A/HK/1/68 reassortants that segment 2, coding for the polymerase-associated protein PB1, and possibly segment 8, encoding the NS1 and NS2 proteins, control the ability of FM-MA to interfere. Interference could not be overcome by increasing the titer of the coinfecting strain, but delaying FM-MA infection by 4 to 6 h did avoid interference. During interference of A/HK/1/68, protein synthesis was inhibited by less than 65% throughout coinfection. Given the kinetics of interference and the small perturbation in protein synthesis, interference appeared to occur at the level of late genome replication or virus assembly. Virulence and interference in FM-MA were not linked. An interfering avirulent FM-MA x A/HK/1/68 reassortant, E07, was capable of protecting mice against lethal pneumonia due to a virulent noninterfering reassortant, H04.     

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

Vero cells, a line of African green monkey kidney cells, failed to produce interferon when infected with Newcastle disease, Sendai, Sindbis, and rubella viruses, although the cells were sensitive to interferon. Further, infection of Vero cells with rubella virus did not result in interference with the replication of echovirus 11, Newcastle disease virus, or vesicular stomatitis virus, even in cultures where virtually every cell was infected with rubella virus. Under the same conditions, BSC-1 cells and other cells of primate origin produced interferon and showed rubella virus interference. The results indicate that the presence of rubella virus in the cell does not in itself exclude multiplication of other viruses and that rubella virus interference appears to be linked to the capability of the cell to produce interferon.     

Viral determinants of the variable sensitivity of herpes simplex virus strains to gD-mediated interference.

Cells that express glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) resist infection by HSV-1 and HSV-2 because of interference with viral penetration. The results presented here show that both HSV-1 and HSV-2 gD can mediate interference and that various HSV-1 and HSV-2 strains differ in sensitivity to this interference. The relative degree of sensitivity was not necessarily dependent on whether the cell expressed the heterologous or homologous form of gD but rather on the properties of the virus. Marker transfer experiments revealed that the allele of gD expressed by the virus was a major determinant of sensitivity to interference. Amino acid substitutions in the most distal part of the gD ectodomain had a major effect, but substitutions solely in the cytoplasmic domain also influenced sensitivity to interference. In addition, evidence was obtained that another viral gene(s) in addition to the one encoding gD can influence sensitivity to interference. The results indicate that HSV-1 and HSV-2 gD share determinants required to mediate interference with infection by HSV of either serotype and that the pathway of HSV entry that is blocked by expression of cell-associated gD can be cleared or bypassed through subtle alterations in virion-associated proteins, particularly gD.     

Homologous Interference by Incomplete Sendai Virus Particles: Changes in Virus-Specific Ribonucleic Acid Synthesis

Incomplete Sendai virus particles (I particles) interfered with the replication of several strains of infectious Sendai virions (standard virus) but not with the replication of Newcastle disease virus, mumps virus, or Sindbis virus. I particles did not induce interferon, and ultraviolet irradiation of I particles abolished their ability to interfere. Protein synthesis was not necessary to establish interference. The degree of interference depended on the interval between exposure of cells to the I particles and challenge by standard virus, and this was reflected in the degree of inhibition of virus-specific ribonucleic acid (RNA) synthesis in infected cells. The most dramatic change was decreased accumulation of 50S virus-specific RNA in infected cells. RNA species sedimenting slower than 50S were not as markedly reduced in total amount, but hybridization experiments showed that a substantial portion of these slowly sedimenting RNA species were plus strands, presumably representing replicas of the RNA species in I particles. When I particles in insufficient numbers to interfere were added to cells as late as 8 hr after standard virus, there were no obvious changes in virus-specific RNA species in the cells; however, significant amounts of 19 and 25S RNA species, representing progeny of the I particles, appeared in the culture medium. It was concluded that interference was an intracellular event affecting an early step in virus replication. Competition by I particles for cell sites or substrates needed by standard virus seemed a less likely mechanism of interference than competition for enzymes specified by standard virus.     

Identification of virus found in mouse lymphomas induced by HIX murine oncornavirus.

Lymphomas induced by pure HIX murine leukemia virus in two mouse strains contained large amounts of virus with amphotropic properties. Analysis of tumor derived virus purified by limiting dilution techniques indicated that its interference and neutralization spectra were essentially identical to parental HIX virus and different from eco- and xenotropic viruses. Examination of virus progeny from many individual foci induced by virus derived directly from lymphomas indicated that each infectious unit contained only HIX virus. No evidence of ecotropic virus presence was observed.     

Distinct superinfection interference properties yet similar receptor utilization by cytopathic and noncytopathic feline leukemia viruses.

Cell killing by cytopathic retroviruses is often associated with a delay or failure in the establishment of superinfection interference. Superinfection has been observed during T-cell killing and fatal immunodeficiency disease induction by the feline leukemia virus (FeLV) chimera FeLV-FAIDS-EECC, containing the surface envelope glycoprotein (SU) of FeLV-FAIDS clone 61C. We demonstrate here that 61C SU has a defect that results in a nearly complete failure to establish superinfection interference against homologous virus challenge. This failure was evident only in feline T (FeT) cell clones expressing envelope protein, not in the rare cells that have survived cytopathic infection to become chronically infected. The regions of SU responsible for this defect were the same as those previously identified as responsible for T-cell killing. The superinfection interference properties of a noncytophatic molecular clone, FeLV-FAIDS-61E, were different in that 61E established interference to homologous virus challenge, both in SU-expressing cell clones and in chronically infected cells. Neither 61E nor EECC established interference against heterologous virus challenge. Viruses expressing chimeric SU proteins displayed varied and intermediate interference properties. Purified 61E and 61C SU competed for binding sites on FeT cell surfaces, and purified 61E SU blocked infection of virus bearing 61E or 61C SU. In addition, purified 61E and 61C SU each coprecipitated 70-kDa FeT cell surface proteins. Our data are consistent with the hypothesis that there are multiple cellular components necessary for 61E and 61C attachment to and penetration of FeT cells, a primary receptor that is utilized by both 61E and 61C, and secondary receptors that are likely to be virus specific.     

Interaction Between Cytomegalovirus and Newcastle Disease Virus as Mediated by Intrinsic Interference

Cytomegalovirus (CMV) was demonstrated to induce intrinsic interference to Newcastle disease virus (NDV) in human fibroblast cells under noncytopathic conditions. This interference is unique in that (i) cytomegalovirus is the first DNA virus demonstrated to have this property and (ii) the state of interference was transient and progressively lost as the condition of the cells changed with the development of cytopathic effect. These observations are consistent with the view that the newly formed protein responsible for interference with NDV has a limited half-life and is no longer made when cytopathic conditions are produced by CMV.     

Interference Among Group A Arboviruses

Interference among group A arboviruses is described which does not involve the mediation of interferon. Interference was observed only if the interfering virus had an advantage over the challenge virus, either in time or in multiplicity of infection. Adsorption, penetration, and uncoating of challenge virus did not appear to be inhibited, but the synthesis of infectious viral ribonucleic acid of the challenge virus was significantly retarded. It was shown with temperature-sensitive viruses or mutants that the replication of viral ribonucleic acid by the interfering virus was required to establish interference. A mechanism of interference based on a competition for replication sites or substrates is compared with other possible explanations.     

Inhibition of virus production in JC virus-infected cells by postinfection RNA interference

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.     

Isolation of an RD-114-Like Oncornavirus from a Cat Cell Line

A clone of cells derived from a continuous line of cat cells (CCC) spontaneously produced an RNA C-type virus (CCC virus) which did not have the group-specific antigen of the standard strains of feline leukemia viruses but did have that of the RD-114 virus. Single-hit infection of a virus yielding CCC cell with only the feline leukemia virus pseudotype of murine sarcoma virus [MSV(FeLV)] resulted in the release of a pseudotype of MSV coated with the CCC virus envelope. Host range, transmission of virus, helper functions, interference properties, and specific neutralization showed that the CCC and the RD-114 isolates as well as their respective MSV pseudotypes are closely similar if not identical. Parental, virus-negative cells frozen before the existence of RD-114 were chemically induced to yield CCC-like virus de novo. Infection of susceptible human cells with the chemically induced virus resulted in interference with the CCC virus pseudotype of MSV but not with the FeLV pseudotype of MSV.     

Insights into RNA virus mutant spectrum and lethal mutagenesis events: replicative interference and complementation by multiple point mutants

RNA virus behavior can be influenced by interactions among viral genomes and their expression products within the mutant spectra of replicating viral quasispecies. Here, we report the extent of interference of specific capsid and polymerase mutants of foot-and-mouth disease virus (FMDV) on replication of wild-type (wt) RNA. The capsid and polymerase mutants chosen for this analysis had been characterized biochemically and structurally. Upon co-electroporation of BHK-21 cells with wt RNA and a tenfold excess of mutant RNA, some mutants displayed strong interference (<10% of progeny production by wt RNA alone), while other mutants did not show detectable interference. The capacity to interfere required an excess of mutant RNA and was associated with intracellular replication, irrespective of the formation of infectious particles by the mutant virus. The extent of interference did not correlate with the known types and number of interactions involving the amino acid residue affected in each mutant. Synergistic interference was observed upon co-electroporation of wt RNA and mixtures of capsid and polymerase mutants. Interference was specific, in that the mutants did not affect expression of encephalomyocarditis virus RNA, and that a two nucleotide insertion mutant of FMDV expressing a truncated polymerase did not exert any detectable interference. The results support the lethal defection model for viral extinction by enhanced mutagenesis, and provide further evidence that the population behavior of highly variable viruses can be influenced strongly by the composition of the quasispecies mutant spectrum as a whole.