[The autointerfering properties of a population of influenza viruses containing defective virus particles].
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Animals↗
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Adeno-associated virus: from defective virus to effective vector.
The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.
Adenoviridae↗
In murine AIDS, B cells are early targets of defective virus and are required for efficient infection and expression of defective virus in T cells and macrophages.
Previous studies showed that B cells and CD4+ T cells are required for induction of a murine retrovirus-induced immunodeficiency syndrome, murine AIDS. Using B6 mice deficient in mature B cells as a result of a knockout of the transmembrane exon of the immunoglobulin M gene, we found that spleen and other tissues from murine AIDS virus-infected mice did not express the defective virus (BM5def) required for induction of disease, even though helper viruses were readily detectable and BM5def proviral DNA was present. This indicates that the B-lineage cells are the primary targets for infection and expression of the defective virus and that in the absence of mature B cells, there is inefficient infection of T cells and macrophages.
Actins↗
Infection of the central nervous system of mice by standard Sendai virus, defective interfering Sendai virus and the mixture of both: comparison of virus multiplication and pathogenicity.
The intracerebral (i.c.) infection of newborn mice with standard Sendai virus (SV), defective interfering Sendai virus (DV) and their mixture (SV + DV) has been used as a model for the possible role of defective interfering particles of paramyxoviruses in several chronic degenerative diseases of central nervous system (CNS). The dynamics of Sendai virus multiplication and virus distribution in CNS of mice, as well as the histological changes and the clinical symptoms were evaluated for up to 112 days post-infection (p.i.). The infectious virus was detected in the brains of animals inoculated i.c. either with SV, or DV, or SV + DV as soon as by 5 hr p.i., with maximum infectivity titre at 24 hr p.i. In brains of animals inoculated with SV, the virus was detected until 5th day p.i.; nevertheless in those, inoculated with SV + DV or DV, low infectious titres could be detected even at later intervals. In mice inoculated i.c. with DV, traces of Sendai virus were detected in subpassages, as late as 3 months p.i.
Animals↗
[Persistance of the genome of the Rous virus in hamster cells converted in vitro by a non defective virus and a defective virus].
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Animals↗
Minireview: the herpes simplex virus amplicon--a versatile defective virus vector.
The Herpes simplex virus (HSV) amplicon was designed in our laboratory, as a defective virus vector, capable of shuttle delivery of DNA sequences and genes from prokaryotic to eukaryotic cells, tissues or organs. The HSV vector was termed "amplicon" to delineate the fact that it carried the cloned sequences of interest, within amplified concatemeric defective genomes, packaged in HSV virions. Employing the replication functions of their helper virus, the amplicons are wide tropic vectors, capable of entry, replication, and gene expression in varied types of cells, both in vitro, and in vivo. In this brief review, the amplicons will be revisited, beginning with studies of the naturally occurring defective genomes, where many of the virologic aspects of the system were established. Properties of the amplicon system and its components, will be enlightened. The cis acting functions required for amplicon propagation will be briefly described. This will follow with examples of our studies of prokaryotic, viral and eukaryotic DNA sequences, which were amplified and expressed within HSV defective genomes. Studies designed to examine the involvement of helper viruses will be described, including our analyses of host shutoff mutant helper viruses. Finally, recent studies of other laboratories will be reviewed, with emphasis on what appears to be the use of amplicons as neurotropic vectors, towards potential gene therapy.
DNA, Viral↗
Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis.
Influenza WSN virus temperature-sensitive (ts) mutants were examined for defects in viral complementary RNA (cRNA) synthesis. The synthesis of viral cRNA was determined by hybridizing RNA from infected cells to radiolabeled virion RNA of known specific activity. Mutants in complementation groups I and III synthesized little, or no, cRNA at the nonpermissive temperature (39.5 C). When cells infected by these mutants were incubated for 5 h at the permissive temperature (33 C) and were then shifted to 39.5 C, net synthesis of cRNA ceased. This strongly suggests that mutants in these two complementation groups possess a ts defect in the transciptase complex. Mutants in group II and group V synthesize reduced amounts of cRNA at 39.5 C. In contrast to the group I and group III mutants, cRNA synthesis in cells infected by a group II or a group V mutant continues after a shift-up. This indicated that these mutants do not possess a ts transcriptase complex and that these mutants are most probably defective in some step in the amplification of cRNA synthesis. As will be discussed, the most likely defect in these mutants is in the synthesis of virion-type RNA. These results suggest that there are two influenza viral gene functions required for transcription and most likely two additional gene functions required for RNA replication.
Cell Line↗
Evidence for specific packaging of the influenza A virus genome from conditionally defective virus particles lacking a polymerase gene.
The incorporation of the eight gene segments of influenza A virus in virions, either through a random or a specific mechanism, has been under debate for many years. Using reverse genetics techniques and trans-complementation of viral proteins, we showed that the production of viruses containing seven gene segments is very inefficient, which can be overcome by providing the 8th gene segment in a non-functional form. We conclude that sequences in the 5' and 3' ends of the polymerase gene segments contain packaging signals. Our methods will facilitate the further mapping of the packaging signals of influenza A virus.
DNA-Directed RNA Polymerases↗
Genetic structure of avian myeloblastosis virus, released from transformed myeloblasts as a defective virus particle.
Chicken myeloblasts transformed by avian myeloblastosis virus (AMV) in the absence of nondefective helper virus (termed nonproducer cells) were found to release a defective virus particle (DVP) that contains avian tumor viral gag proteins but lacks envelope glycoprotein and a DNA polymerase. Nonproducer cells contain a Pr76 gag precursor protein and also a protein that is indistinguishable from the Pr180 gag-pol protein of nondefective viruses. The RNA of the DVP is 7.5 kilobases (kb) long and is 0.7 kb shorter than the 8.2-kb RNAs of the helper viruses of AMV, MAV-1 and MAV-2. Comparisons based on RNA.cDNA hybridization and mapping of RNase T1-resistant oligonucleotides indicated that DVP RNA shares with MAV RNAs nearly isogenic 5'-terminal gag and pol-related sequences of 5.3 kb and a 3'-terminal c-region of 0.7 kb that is different from that found in other avian tumor viruses. Adjacent to the c-region, DVP RNA contains a contiguous specific sequence of 1.5 kb defined by 14 specific oligonucleotides. Except for two of these oligonucleotides that map at its 5' end, this sequence is unrelated to any sequences of nondefective avian tumor viruses of four different envelope subgroups as well as to the specific sequences of fibroblast-transforming avian acute leukemia and sarcoma viruses of four different RNA subgroups. The specific sequence of the DVP RNA is present in infectious stocks of AMV from this and other laboratories in an AMV-transformed myeloblast line from another laboratory, and it is about 70% related to nucleotide sequences of E26 virus, an independent isolate of an AMV-like virus. Preliminary experiments show DVP to be leukemogenic if fused into susceptible cells in the presence of helper virus. We conclude that DVP RNA is the leukemogenic component of infectious AMV and that its specific sequence, termed AMV, may carry genetic information for oncogenicity. Thus we have found here a transformation-specific RNA sequence, unrelated to helper virus, in a highly oncogenic virus that does not transform fibroblasts.
Animals↗
Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes.
Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting of head-to-tail reiterations of sequences derived from the end of the S component of the standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to Mr 100 X 10(6) DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA.
Animals↗
[Delta virus: now also detected in Austria. A defective virus as a pathogenic agent].
delta-virus, first described by Rizzetto, is a defective virus dependent on hepatitis B virus as helper-virus. Superinfection of inactive hepatitis B with delta-virus leads to chronic active or even fulminant hepatitis. It can be detected by RIA or ELISA and its distribution is world-wide. Also in Austria, 4 sera with antibodies to delta-virus have been found among 138 HBsAg-positive sera. No therapy is known, but vaccination against HB virus also prevents the propagation of delta-virus.
Austria↗
Deletions of specific regions of the simian sarcoma-associated virus genome are found in defective viruses and in the simian sarcoma virus.
Extrachromosomal DNA was isolated from tissue culture cells that were acutely infected with simian sarcoma virus (SSV) and its associated helper (simian sarcoma-associated virus [SSAV]). Two sizes of closed circular viral genomic DNA intermediates were isolated, cleaved at the single EcoRI site, and ligated to the Charon 21A phage lambda vector. Cloned molecules of the larger size all represented the full-length (9.0-kilobase [kb]) SSAV molecule. A heterogeneous group of clones was derived from the smaller DNA circles. These included the SSV genome and SSAV deletion mutants. When two SSV clones were compared with the helper, they contained the following three characteristic deletions: (i) a 250-base pair deletion in the gag gene about 1.0 kb from the 5' end of the genome; (ii) a 1.85-kb deletion in the pol gene; and (iii) a 1.9-kb deletion at the 3' end, which included part of the env gene. This latter deletion was the site of the onc gene substitution. Six other clones of the smaller molecules represented the following variants of the SSAV genome: (i) two clones of the entire genome containing only one long terminal repeat unit; (ii) one clone with the 1.85-kb deletion of the pol gene observed in SSSV; and (iii) three clones having a deletion of the 3' end of the SSAV genome. In each of the latter clones, the 5' border of the deletion was indistinguishable from the 5' border of the onc substitution in SSV. The fidelity of genetic deletions observed suggested that certain regions of the SSAV genome were deleted at a high frequency. In certain cases, these deletions may have been accompanied by a substitution of cellular sequences to generate SSV.
Chromosome Deletion↗
Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells.
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Animals↗
Hepatitis delta virus. No longer a defective virus.
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Hepatitis B↗
Presence of an encephalomyocarditis virus internal ribosome entry site sequence in avian infectious bronchitis virus defective RNAs abolishes rescue by helper virus.
Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system. The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette. However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes. The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system. IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation. CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase. However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV. Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.
Animals↗
Generation of defective virus after infection of newborn rats with reovirus.
When 2-day-old rats were inoculated subcutaneously with the R2 strain of reovirus type 3 or with a class B (352) or class C (447) temperature-sensitive (ts) mutant, 5 to 10% of the animals died from acute encephalitis within 12 days. Approximately half of the survivors recovered rapidly and grew normally, but the remainder became runted. Two phases of infection are distinguished in the animals: an acute phase during which infectious virus reaches a maximum titer in brain and other tissues by 10 days p.i. and thes runting of the rats and the slow disappearance of virus from their brains over a period of 2 months or so. Virus isolated from chronically infected brains generally retained the genetic character (ts or wild type) of the inoculated virus, but two exceptions to this are described. Defective virions lacking the L1 segment of the viral genome (L1 defectives) were generated in rat brains during the acute phase of infection. Defective virus was also generated during the chronic phase, but during this period defectives were found with multiple segments deleted from the genome in addition to L1 defectives. In another type of experiment defective virus exerted a marked protective effect when inoculated intracerebrally with R2 virus. In the absence of defectives all animals died, but in their presence 17 of 23 animals survived and 15 of 23 became runted and chronically infected. The formation and evolution of defective particles in the brains of these rats were similar to those found in rats chronically infected after subcutaneous inoculation of reovirus. We conclude that the formation of defective virus particles may play a role in the initiation and maintenance of chronic neutropic infections with reovirus.
Animals↗
The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector.
We have employed repeat units of herpes simplex virus (HSV) defective genomes to derive a cloning-amplifying vector (amplicon) that can replicate in eucaryotic cells in the presence of standard HSV helper virus. The design of the HSV amplicon system is based on the previous observation that cotransfection of cells with helper virus DNA and seed monomeric repeat units of HSV defective genomes results in the regeneration of concatemeric defective genomes composed of multiple reiterations of the seed repeats. Cotransfection of cells with helper virus DNA and chimeric repeat units containing bacterial plasmid pKC7 DNA resulted in the generation of defective genomes composed of reiterations of the seed HSV-pKC7 repeats. These chimeric defective genomes were packaged into virus particles and could be propagated in virus stocks, with the most enriched passages containing more than 90% chimeric defective genomes. Furthermore, monomeric chimeric repeat units could be transferred back and forth between bacteria and eucaryotic cells. A derivative vector constructed so as to contain several unique restriction enzyme sites could be potentially employed in the introduction of additional viral or eucaryotic DNA sequences into eucaryotic cells.
Animals↗
Replication-defective viruses modulate immune responses.
By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.
Animals↗