Satellite viruses, helper viruses, and synergistic action.
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Adenoviridae↗
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Satellite viruses, helper viruses, and synergistic action--discussion.
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Adenoviridae↗
Abelson murine leukemia virus: effect of helper virus from regressing Friend virus on leukemia development.
Replication defective Abelson murine leukemia virus (A-MuLV) induces a non-thymic lymphoma in vivo and transforms both hematopoietic and fibroblastic cells in vitro. In vivo leukemogenicity and the efficiency of in vitro transformation of hematopoietic cells by A-MuLV are known to be affected by the replication competent helper virus present in A-MuLV stocks. The helper virus isolated from the regressing strain of Friend virus (RF-MuLV) is responsible for the spontaneous regression of erythroleukemia induced by replication defective spleen-focus forming virus and itself induces a lymphocytic leukemia which spontaneously regresses. The diseases produced by A-MuLV stocks containing either RF-MuLV or Moloney leukemia virus, the helper virus associated with the original isolate of A-MuLV, were compared to determine if RF-MuLV can influence the disease produced by a replication defective virus with a discrete transforming gene. Both virus stocks induced leukemias with similar efficiency and gross pathology. Spontaneous regression was not observed when RF-MuLV was used as the helper virus. Examination of the leukemic cells and cell lines derived from leukemic tissues indicated that the target cell for A-MuLV transformation was not affected by the helper virus. Both transformed lymphoid and monocytic cells were cultured from leukemic tissues and established as cell lines. The lymphoid cells were phenotypically similar to pre-B cells or null cells, while the monocytic cell lines resemble promonocytes. The frequency with which promonocytic cell lines were isolated from leukemic mice suggests that A-MuLV leukemogenesis may often involve transformation of monocytic series cells as well as lymphoid cells. Thus, RF-MuLV can serve as an efficient helper virus for A-MuLV and does not appear to alter the in vivo target cell for transformation. It is unable, however, to alter the progressive course of Abelson virus induced disease.
Abelson murine leukemia virus↗
Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus.
A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.
Animals↗
Specificity in the association of tomato black ring virus satellite RNA with helper virus.
The satellite RNAs (sat-RNAs) associated with some isolates of tomato black ring virus (TBRV) consist of single-stranded molecules of about 1375 nucleotides, encoding a nonstructural protein of 48K which has been shown to be involved in the replication of the sat-RNA. The TBRV sat-RNAs are also dependent for their replication and for their encapsidation on the helper virus. To characterize the nature of the association between sat-RNA and helper virus, transcripts of sat-RNA from TBRV isolates C and L (respectively, of serotypes G and S) have been prepared and inoculated onto Chenopodium quinoa leaves or protoplasts. Transcript of the TBRV sat-RNA C is efficiently multiplied when coinoculated with the genomic RNAs of TBRV isolate G (used instead of TBRV isolate C, because isolate G was depleted of sat-RNA), but does not multiply with TBRV isolate L. On the other hand, transcript of the sat-RNA L is able to multiply with the cognate helper virus and, less efficiently, with grapevine chrome mosaic virus (another nepovirus, 80% similar to TBRV), but does not multiply with TBRV G. The specificity of the association resides at the level of sat-RNA replication. Analysis of the multiplication of chimeric sat-RNAs, obtained by exchanging different regions between the two sat-RNAs C and L, showed that the 5' and the 3' noncoding regions of the sat-RNA, although important for replication, are not implicated in specificity. The results suggest that the determinants of the specificity are contained in the 48K sat-RNA-encoded protein.
Base Sequence↗
Sites of recombination between the transforming gene of avian myeloblastosis virus and its helper virus.
The sites of recombination between the transforming gene of avian myeloblastosis virus (AMV) and its natural helper myeloblastosis-associated virus (MAV) have been determined. In AMV, the cellular sequence substituting for the viral envelope (env) gene gives rise to a different carboxyl terminus of the DNA polymerase. The 5'-recombination site coincides with the RNA splice acceptor site for the production of env mRNA in MAV-infected cells. The 3'-recombination site reveals that the last 11 amino acids including the termination codon are shared by the env protein and AMV transforming protein. The RNA splice acceptor site for the generation of subgenomic v-myb mRNA is located 84 nucleotides downstream from the 5'-recombination site. The AMV transforming protein consists of helper virus-related sequences at both of its amino and carboxyl termini, and all but 84 nucleotides of the cell-derived v-myb sequence. The comparison of MAV gp85 amino acid sequence with those of subgroups B, C, and E indicates that the MAV present in clone lambda 10A2-1 belongs to subgroup B. The high degree of homology among different avian retroviruses of the same subgroup indicates that the amino acid sequence of gp85 is important in determining the conformation of the envelope glycoprotein.
Amino Acid Sequence↗
Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.
Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.
Adenoviridae↗
The relationship between genomic RNAs of polycythaemic forms of spleen focus-forming Friend virus and its helper virus.
We have studied the relationship between Friend spleen focus-forming virus (SFFV) and its helper lymphoid leukaemia virus (LLV) by comparing RNase T1 fingerprints of genomic RNAs. Our data indicate that about 70% of the SFFV sequence is a perfect copy of parts of the helper genome. We conclude that our SFFV and LLV isolates have co-evolved very closely and that SFFV-specific sequences are not identical in different Friend virus isolates.
Base Sequence↗
Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors.
Helper-dependent adenovirus vectors (hdAd), which are deleted of all viral protein-coding sequences, can mediate long-term expression of a therapeutic transgene and lead to life-long, phenotypic correction in animal models of genetic disease. Here, we describe a new system for the generation of hdAd, which utilizes the DNA size restrictions imposed on an Ad virion deleted of protein IX (pIX): such virions are reported to package up to only approximately 35 kb of viral DNA. A pIX(-) helper virus (approximately 37.3 kb) was easily grown on complementing 293pIX cells. Upon infection of noncomplementing cells, this virus was not capable of forming infectious virions, but provided replicative and packaging functions for propagation of a 30-kb hdAd. The pIX(-) helper virus was effective in amplifying an hdAd and, in combination with Cre-mediated excision in the viral-packaging signal, resulted in a 1000-fold reduction in helper virus contamination in hdAd stocks compared to Cre/lox alone, as determined by plaque assay. However, through slot blot analysis of DNA isolated from virions, we determined that the ratio of hdAd to helper DNA was 500:1, similar to the ratio observed when using Cre/lox alone. Surprisingly, a large amount of the 37.3-kb helper DNA was being packaged into the pIX-deleted virions, but these virions were incapable of establishing productive infections in plaque assays, for reasons which are still unclear. Nevertheless, the pIX(-) hdAd generated in this system infected cells and expressed a transgene at levels similar to those obtained with a pIX(+) hdAd. These data suggest that, although further studies are necessary to characterize the nature of the defective helper virions formed in this system, deletion of pIX from the helper virus genome does provide an effective method to prevent recovery of functional helper virus during hdAd amplification.
Adenoviridae↗
Transfection of fibroblasts by cloned Abelson murine leukemia virus DNA and recovery of transmissible virus by recombination with helper virus.
A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.
Abelson murine leukemia virus↗
[Aphid-transmissible viruses and the helper viruses (author's transl)].
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Animals↗
Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses.
BACKGROUND: Helper-dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high-titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo. METHODS: Replication-defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7-cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre-terminal protein, and with a cre-recombinase plasmid. RESULTS: We show that C7-cre cells allow efficient production of gutted Ad using deltaE1 + deltaE2b + deltaE3 helper viruses whose growth can be limited by cre-loxP-mediated excision of the packaging signal. Gutted Ad vectors carrying approximately 28 kb cassettes expressing full-length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%. CONCLUSIONS: These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b-deleted vectors display a profound reduction in viral gene expression.
Adenoviridae↗
Identity of the 5'-terminal RNA nucleotide sequence of the satellite tobacco necrosis virus and its helper virus: possible role of the 5'-terminus in the recognition by virus-specific RNA replicase.
A pancreatic ribonuclease digest of (14)C-labeled tobacco necrosis virus RNA was fractionated according to charge by column chromatography. Individual fractions were dephosphorylated with alkaline phosphatase and rechromatographed. The fraction, originally containing oligonucleotides with seven negative charges, separated into two components corresponding to five (-5) and two negative charges (-2). The -5 fraction was derived from the internal oligonucleotides while the -2 fraction must have originated from a 5'-pyrophosphorylated terminal trinucleotide. The sequence of this terminal trinucleotide was determined by column chromatography on DEAE-cellulose in a triethyl ammonium carbonate gradient, using the appropriate markers. The radioactivity chromatographed with a (ApGp)U marker. The order of the Ap and Gp was determined after ribonuclease T(1) and alkaline phosphatase digestion. The radioactivity in the product chromatographed with an ApG marker. The 5'-terminus of tobacco necrosis virus RNA was therefore determined as ppApGpUp..., which is identical to the terminus of the RNA of its satellite virus as previously determined (J. Mol. Biol., 38, 59 (1968); Science, 160, 1452 (1968)). The 5' pyrophosphate in both viruses was probably formed by an in vivo enzymatic removal of a gamma-phosphate from a triphosphate, and its presence in both viruses suggested a common site of synthesis. The identity of the 5'-terminal sequences is considered not to be fortuitous and is discussed from the standpoint of their role as a recognition site for the virus-specific RNA replicase.
Carbon Isotopes↗
Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus.
Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.
Bacteriophage lambda↗
Cell transformation and tumor induction by Abelson murine leukemia virus in the absence of helper virus.
We investigated the role of the Moloney helper virus, Moloney murine leukemia virus (Mo-MuLV), in cell transformation and tumor induction by the defective Abelson murine leukemia virus (Ab-MuLV). A molecular clone of Ab-MuLV (P160 strain) was transfected into the psi 2 packaging cell line, and helper virus-free Ab-MuLV (psi 2) was harvested from the supernatant medium. Ab-MuLV (psi 2) was as efficient as helper virus-containing Ab-MuLV (Mo-MuLV) in the transformation of primary bone marrow cells in vitro. Inoculation of weanling BALB/c mice with Ab-MuLV (psi 2) induced nonthymic pre-B-cell lymphomas with high efficiency and short latency (28 days). Adult BALB/c mice were less sensitive to tumor induction by a factor of 100. Ab-MuLV (psi 2) did not induce tumors in weanling C57BL/6 mice, unlike Ab-MuLV (Mo-MuLV). Examination of the proviral integration pattern in Ab-MuLV (psi 2)-induced tumor cell DNA revealed that each of the tumors contained a single integrated provirus. Immunoprecipitation of viral-encoded proteins in helper virus-free tumor cell lines detected the P160 Ab-MuLV-transforming protein; however, no trace of the gag, pol, and env helper virus-encoded proteins was found. Our results indicate that integration and expression of a single Ab-MuLV genome is sufficient for efficient transformation of primary bone marrow cells by Ab-MuLV in vitro and tumor induction in susceptible mice. However, the helper virus may contribute to tumor induction in weanling resistant mice.
Animals↗
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↗
Transformed cell lines derived from progressor Sarcoma virus (Moloney) induced tumors. I. Differences in pathogenicity between C type viruses from producer and helper virus infected nonproducer cells.
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Animals↗
A novel approach for herpes simplex virus type 1 amplicon vector production, using the Cre-loxP recombination system to remove helper virus.
Helper-dependent HSV vectors (commonly known as HSV amplicons) are able to transfer genes into both dividing and quiescent cells, and thus have the potential to be widely used as vectors in physiological studies and gene therapy. Historically, these vectors were produced by superinfection with a helper virus that furnished all the trans-acting functions required for amplification and packaging of vector genomes into HSV-1 particles. In these systems, however, large amounts of potentially harmful helper virus are present in the vector stocks, thus restricting the use of these vectors. New helper virus-free packaging systems have been developed that utilize transfection of helper functions rather than infection and thus produce safer vector stocks. The vector titers as well as the amounts of particles obtained with these systems are, however, limited by the impossibility to reamplify the vector stocks. In this article, we present a novel system for producing large amounts of high-titer amplicon vector with low contamination by helper viruses. This system is based on the use of the Cre-loxP recombination system, which allows efficient deletion of the packaging signal of an HSV-1 recombinant helper virus (HSV-1-LaL) on Cre-expressing cells (TE-CRE30).
Animals↗