A method for helper virus-free production of adeno-associated virus vectors.
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Helper virus-dependent replication, nucleotide sequence and genome organization of the satellite virus of maize white line mosaic virus.
Virus like particles (17 nm in diameter) associated with maize white line mosaic virus (MWLMV) were shown to be a satellite virus of MWLMV (SV-MWLMV) on the basis of the following properties: (1) The SV-MWLMV was dependent upon the presence of MWLMV for replication in maize, while the latter virus could replicate independently of the SV particles. (2) No nucleotide sequence homology was detected between the SV-MWLMV and MWLMV, using complementary DNA probes prepared to the two RNAs, in a Northern blot hybridization analysis. (3) The RNA of the SV-MWLMV translated in vitro to produce a protein of the same Mr (24,000) as that found associated with the SV particles. This protein could be immunoprecipitated with an antiserum to the SV particles. And (4), there was no serological relationship between the coat proteins of MWLMV and the SV-MWLMV. The complete nucleotide sequence of the SV-MWLMV RNA (1168 nucleotides) was determined. The SV-MWLMV RNA contains a single open reading frame encoding a polypeptide of Mr 23,961. Computer analysis revealed no significant homology between SV-MWLMV RNA and any other viral or satellite RNAs. However, the putative SV-MWLMV capsid protein is predicted to share some structural features with the capsid protein of the satellite virus of panicum mosaic virus.
Amino Acid Sequence↗
Structure and specific sequences of avian erythroblastosis virus RNA: evidence for multiple classes of transforming genes among avian tumor viruses.
Two major RNA species were found in several clonal isolates of avian erythroblastosis virus (AEV) and avian erythroblastosis-associated helper virus (AEAV) complexes: one of 8.7 kilobases (kb), the other of 5.5 kb. The 5.5-kb species was identified as AEV RNA because (i) it was absent from non-transforming AEAV isolated from the same virus complex, (ii) it was present in complexes of AEV and different helper viruses, and (iii) its structure is similar to that of avian acute leukemia viruses of the MC29 group. Molecular hybridization indicated that 54% of AEV RNA is specific and 46% is related to other viruses of the avian tumor virus group, particularly to AEAV, therefore termed group-specific. The genetic structure of AEV RNA was deduced by mapping oligonucleotides representing specific and group-specific sequences and by comparing the resulting map to maps of AEAV and of other avian tumor viruses derived previously. AEV RNA contains a gag gene-related, 5' group-specific section of 1 kb, an internal AEV-specific section of 3 kb unrelated to any other viral RNA tested, and a 3' group-specific section of 1.5 kb. The 5' section of AEV RNA is closely related to analogous 5' sections of the MC29 group viruses and is homologous with a 5' RNA section that is part of the gag gene of AEAV. The 3' section is also shared with AEAV RNA and includes a variant C-oligonucleotide near the 3' end that is different from the highly conserved counterparts of all other exogenous avian tumor viruses. By analogy with Rous sarcoma virus and the acute leukemia viruses of the MC29 group, the internal specific section of AEV RNA is thought to signal a third class of onc genes in avian tumor viruses. Comparisons with AEAV and the MC29 group viruses suggest that both the 5' gag-related and the internal specific RNA sections of AEV are necessary for onc gene function.
Alpharetrovirus↗
Nucleotide sequence of the transforming gene of avian myeloblastosis virus.
Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.
Animals↗
High-frequency leader sequence switching during coronavirus defective interfering RNA replication.
A system was developed that exploited defective interfering (DI) RNAs of coronavirus to study the role of free leader RNA in RNA replication. A cDNA copy of mouse hepatitis virus DI RNA was placed downstream of the T7 RNA polymerase promoter to generate DI RNAs capable of extremely efficient replication in the presence of a helper virus. We demonstrated that, in the DI RNA-transfected cells, the leader sequence of these DI RNAs was switched to that of the helper virus during one round of replication. This high-frequency leader sequence exchange was not observed if a nine-nucleotide stretch of sequence (UUUAUAAAC) at the junction between the leader and the remaining DI sequence was deleted. This observation suggests that a free leader RNA generated from the genomic RNA of mouse hepatitis virus may participate in the replication of DI RNA.
Base Sequence↗
New mammalian transforming retrovirus: demonstration of a polyprotein gene product.
A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.
Animals↗
ras induced lesions in a heterotopic mouse bladder.
To determine the in vivo phenotype elicited in bladder epithelium following the expression of a ras oncogene we have introduced the HaSV ras transforming gene into transplants of normal urothelium. Stripped adult bladder mucosa incubated with HaSV, in the presence or absence of helper virus, was transplanted beneath the renal capsule of syngeneic animals and maintained for periods up to three months. Control implants, exposed to helper virus alone, formed heterotopic bladders lined by urothelium displaying focal areas of full differentiation when associated with underlying submucosal elements. Exposure of urothelium to HaSV resulted in increased proliferative potential of mucosal and submucosal elements with the appearance of a normal differentiated heterotopic bladder 10 days post-implantation. Implants left for 28 days presented a range of hyperplastic lesions (mild-severe) characterized from histological evaluation and antibody markers recognizing basal (D66) and superficial cell populations (H10) in normal bladder mucosa. Staining of mild hyperplastic lesions revealed an increased basal cell compartment and a loss of fully differentiated cells lining the lumen of the implanted bladder. In severe hyperplasia no superficial cells were observed but mucosal elements stained throughout with antibody D66. This phenotype was accompanied by an irregular laminin staining pattern associated with a disorganized basement membrane and increased blood vasculature. Similar experiments conducted with HaSV and helper virus resulted in the generation of mesenchymal lesions at 28 days with little surviving urothelium. The H-ras oncogenic protein can induce hyperplastic lesions in normal urothelium which were characteristic of preneoplastic changes identified in bladder carcinogenesis.
Animals↗
Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences.
Previous studies (R. R. Spaete and N. Frenkel, Cell 30:295-304, 1982) have documented the potential use of defective virus vectors (amplicons) derived from herpes simplex virus for the efficient introduction of foreign DNA sequences into eucaryotic cells. Specifically, cotransfection of cells with helper virus DNA and cloned amplicons (8 to 10 kilobases [kb]) containing bacterial plasmid DNA sequences linked to a set of herpes simplex virus cis-acting propagation signals (a replication origin and a cleavage-packaging signal) resulted in the generation of virus stocks containing packaged defective genomes that consisted of uniform head-to-tail reiterations of the chimeric seed amplicon sequences. The chimeric defective genomes could be stably propagated in virus stocks and could thus be used to efficiently infect cells. We now report on additional studies designed to propagate relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. These studies have utilized a 12-kb chicken DNA sequence encoding the chicken ovalbumin gene and cloned by Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 77:244-248, 1980) in the plasmid pOV12. Virus stocks derived from cells cotransfected with helper virus DNA and chimeric amplicons (overall size of 19.8 kb, of which 12 kb corresponded to the chicken DNA) contained defective genomes composed of reiterations of the 19.8-kb seed amplicon sequences. However, in addition to the authentically sized repeat units, defective genomes in the derivative virus stocks contained smaller repeat units representing deleted versions of the seed 19.8-kb amplicons. The recombinational events leading to the formation of deleted repeats did not appear to occur at unique sites, as shown by comparative analyses of multiple, independently generated virus series propagated from separate transfections. In contast, seed amplicons ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences replicated efficiently and could be stably propagated in virus stocks. The results of these studies suggest the existence of size restrictions (up to 15 kb) on the efficient replication of seed herpes simplex virus amplicons.
Animals↗
Oligoribonucleotide map and protein of CMII: detection of conserved and nonconserved genetic elements in avian acute leukemia viruses CMII, MC29, and MH2.
RNA and protein of the defective avian acute leukemia virus CMII, which causes myelocytomas in chickens, and of CMII-associated helper virus (CMIIAV) were investigated. The RNA of CMII measured 6 kilobases (kb) and that of CMIIAV measured 8.5 kb. By comparing more than 20 mapped oligonucleotides of CMII RNA with mapped and nonmapped oligonucleotides of acute leukemia viruses MC29 and MH2 and with mapped oligonucleotides of CMIIAV and other nondefective avian tumor viruses, three segments were distinguished in the oligonucleotide map of CMII RNA: (i) a 5' group-specific segment of 1.5 kb which was conserved among CMII, MC29, and MH2 and also homologous with gag-related oligonucleotides of CMIIAV and other helper viruses (hence, group specific); (ii) an internal segment of 2 kb which was conserved specifically among CMII, MC29, and MH2 and whose presence in CMII lends new support to the view that this class of genetic elements is essential for oncogenicity, because it was absent from an otherwise isogenic, nontransforming helper, CMIIAV; and (iii) a 3' group-specific segment of 2.5 kb which shared 13 of 14 oligonucleotides with CMIIAV and included env oligonucleotides of other nondefective viruses of the avian tumor virus group (hence, group specific). This segment and analogous map segments of MC29 and MH2 were not conserved at the level of shared oligonucleotides. CMII-transformed cells contained a nonstructural, gag gene-related protein of 90,000 daltons, distinguished by its size from 110,000-daltom MC29 and 100,000-dalton MH2 counterparts. The gag relatedness and similarity to the 110,000-dalton MC29 counterpart indicated that the 90,000-dalton CMII protein is translated from the 5' and internal segments of CMII RNA. The existence of conserved 5' and internal RNA segments and conserved nonstructural protein products in CMII, MC29, and MH2 indicates that these viruses belong to a related group, termed here the MC29 group. Viruses of the MC29 group differ from one another mainly in their 3' RNA segments and in minor variations of their conserved RNA segments as well as by strain-specific size markers of their gag-related proteins. Because (i) the conserved 5' gag-related and internal RNA segments and their gag-related, nonvirion protein products correlate with the conserved oncogenic spectra of the MC29 group of viruses and because (ii) the internal RNA sequences and nonvirion proteins are not found in nondefective viruses, we propose that the conserved RNA and protein elements are necessary for oncogenicity and probably are the onc gene products of the MC29 group of viruses.
Animals↗
Mutant avian erythroblastosis virus with restricted target cell specificity.
Avian erythroblastosis virus (AEV) induces a fatal erythroblastosis within 2 weeks of intravenous injection in chicks in virtually 100% of cases. In chicks injected intramuscularly, sarcomas frequently develop at the site of injection before the animals die from erythroblastosis. In vitro, AEV transforms both erythroblasts, derived from bone marrow cultures, and fibroblasts. These effects have been shown to be a general property of AEV and not of separate leukaemia- and sarcoma-inducing forms of the virus. AEV is defective for replication and can be propagated only in the prewence of helper virus. Its transformation specificity is independent of the helper virus used. It is not clear whether AEV has two different genes controlling transformation of the two types of target cell or whether it has only one gene coding for both. To investigate this question, we looked for mutants of AEV unable to transform one of the two types of target cell. We now describe such a mutant, which is defective for erythroblast transformation but which can still transform fibroblasts.
Alpharetrovirus↗
Artificial defective interfering RNAs derived from brome mosaic virus.
Naturally occurring defective interfering RNAs (DI-RNAs) greatly reduce the accumulation of their helper virus in vivo, but are rarely associated with plant positive-strand RNA viruses. Deletion mutants pRNA-2 M/S and pRNA-2 E/S, derived from brome mosaic virus (BMV) genomic RNA-2, replicated in a manner dependent on BMV RNA-1 and -2, and effectively interfered with their accumulation in barley protoplasts. Based on their mode of replication, these mutant RNAs have been termed parasitic RNAs (pRNAs). When present with RNA-1 and -2 at low inoculum amounts, pRNA-2 M/S and pRNA-2 E/S reduced the level of replication of RNA-2, the parental RNA, by 37% and 64%, respectively. Greater amounts of pRNA in the inoculum completely eliminated the replication of both RNA-1 and -2. Mutations that prevented translation of truncated proteins from the pRNAs did not affect interference, but those that reduced pRNA replication decreased their ability to interfere with genomic RNA replication. At a molar pRNA: genomic RNA inoculum ratio of 1.5:1, pRNA-2 E/S reduced the accumulation of all helper virus RNAs by greater than 60%. This occurred in the presence of wild-type RNA-3 or delta SGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, demonstrating that the interference mediated by the pRNAs was not effected by encapsidation. These data indicate that the expression of pRNAs that function as artificial DI-RNAs in transgenic plants may be an approach for inducing resistance to virus infection which is applicable to a wide range of plant viruses.
Blotting, Northern↗
HHV-6 and HHV-7 as exogenous agents in human lymphocytes.
Human herpesviruses 6 and 7 (HHV-6 and HHV-7), the newly recognized lymphotropic members of the herpesvirus family, were isolated from peripheral blood mononuclear cells (PBMC) of healthy individuals. HHV-7 was recovered from PBMC after exposing the cells to conditions leading to T cell activation. No virus could be recovered from quiescent non-activated cultures, suggesting that the virus resided latently in these cells and that it could be induced from latency by T cell activation. HHV-6 could not be recovered from PBMC using similar approaches. However, it could be repeatedly reactivated from PBMC after infection of the cells with HHV-7. Upon further passaging in culture, HHV-6 took over the population and virus stocks appeared to be clear of the original HHV-7 helper virus. The results of this study suggest that genomes of the lymphotropic herpesviruses are resident in PBMC of healthy adults. HHV-7 can supply the functions required for its reactivation from latency. It can also act as a helper virus for the reactivation of HHV-6 from latency. Following the initial reactivation step, HHV-6 can replicate autonomously in the cells. These results may be relevant to the findings that HHV-6 infection is induced in immunosuppressed patients and in patients undergoing organ transplantation. Furthermore, these studies may exemplify situations whereby propagation of known agent(s) in vitro results in the activation of human pathogens resident latently in the cells.
Adult↗
A novel subviral agent associated with a geminivirus: the first report of a DNA satellite.
Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem-loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.
Base Sequence↗
Isolation of two transforming viruses from sarcomas obtained in chickens inoculated intraembryonally with a transformation defective mutant of Prague strain Rous sarcoma virus.
Sarcomas appeared after long latency with a frequency of about 2% in Brown Leghorn and (CB X IC)F1 chickens after intraembryonic and neonatal inoculation of transformation-defective mutants of ASVs subgroup C. Only the freshly isolated td mutants, td daPR-C and td daPR-C morphf, exhibited the tumorigenic activity, whereas the standard td mutants induced no sarcomas. Two (862 and 2257) out of four tumours could be transplanted in young chickens and produced low titres of transforming virus. The other two tumours were not transplantable and devoid of any transforming virus. Viruses 862 and 2257 are clearly defective in replication, virus 2257 cannot be complemented efficiently by any helper virus in vitro. The low titre of virus 2257 is not caused by interference with a helper virus of the same subgroup specificity. Both viruses are highly tumorigenic in vivo.
Animals↗
The history of the HSV amplicon: from naturally occurring defective genomes to engineered amplicon vectors.
We have derived the HSV amplicon vector in 1981/1982 after elaborate experience with "defective viruses", arising spontaneously in viral stocks propagated at high multiplicities of infection (m.o.i.). The defective viruses were found to contain large concatemeric genomes with repeat units of limited complexity. We employed cloned defective genome repeats to generate the "amplicon" vectors, which in the presence of helper virus replicate to produce packaged large concatemeric genomes, transmissible to uninfected cells. The cloned amplicons were then employed to fine map and analyze the signals essential for amplicon propagation: (i) A DNA replication origin, producing concatemeric genomes by rolling circle replication. Three DNA replication origins were identified in the HSV genome. (ii) Signals termed pac-1 and pac-2, directing a measuring function for coordinate cleavage of the concatemeric genomes and their packaging as full-size (150 kb) genomes. Using amplicons, foreign genes of large sizes could be linked to less than 1 kb of the cis-acting HSV DNA sequences and become amplified in packaged defective genomes, transmissible to new cells. The transgenes are expressed efficiently, due to sequence reiterations. Large quantities of vectors can be produced in vitro. The amplicons are attractive vectors for use as non-integrating gene delivery vectors. The packaging signals pac-1 and pac-2 are well conserved in different herpesviruses and amplicons with a DNA replication origin and cleavage and packaging signals have been produced in additional herpesviruses. Depending on amplicon-host cell combination, the vectors can be employed with and without mutated helper virus(es) to obtain high gene expression, and desired effect on the target cell. In the absence of helper virus, the defective virus produced is limited for spread in the targeted cells. We expect that new vectors employing state of the art transgenes, will be developed to generate amplicon based concatemeric defective viruses capable of efficient expression of these genes.
Base Sequence↗
Thymocyte subsets transformed by Abelson murine leukemia virus.
The infectious complex of Abelson murine leukemia virus was altered by replacing its usual helper virus, Moloney leukemia virus, with radiation leukemia virus (RadLV). After intrathymic injection of the Abelson-RadLV complex, thymomas arose rapidly, as described previously for injection of the Abelson-Moloney complex. Cell lines were derived from thymomas induced by each Abelson virus complex and were classified according to normal thymus cell phenotypes. Each virus complex induced some cell lines which were like a 0.7% subpopulation of murine thymocytes in that they failed to express the Thy-1 cell-surface antigen. These lines are thus far indistinguishable from some Abelson-derived bone marrow transformants classified as pre-B cells. However, the Abelson-Moloney complex induced some cell lines which expressed low levels of Thy-1 and which shared most markers with immature blast cells of the thymic medulla, whereas the Abelson-RadLV complex induced some lines which were clearly like thymic cortex blast cells. Thus, Abelson virus can induce thymoma cell lines of at least two, and possibly three, distinct phenotypes corresponding to normal thymocyte blast subsets, the determination of which can be influenced by helper virus sequences.
Abelson murine leukemia virus↗
[Significance of priming of hosts with virus in the tumor-specific immunotherapy model utilizing virus-reactive helper T cell activity].
A tumor-specific immunotherapy model was established utilizing vaccinia virus (VV) in the consideration of clinical application, in which we assessed the significance of priming of hosts with VV to induce in vivo anti-tumor immunity. C3H/HeN mice were pretreated with cyclophosphamide (Cy) to eliminate non-specific suppressor T cell activity and subsequently inoculated (primed) subcutaneously (s.c.) with 10(7)PFU of VV, leading to augmented induction of VV-reactive helper T (VV-Th) cell activities. Four weeks later, the mice were inoculated intradermally (i.d.) with syngeneic X5563 myeloma cells. Six days after the tumor cell inoculation, 5 X 10(7)PFU of VV was injected into the tumor mass 3 times at 2-day intervals. Seven of 10 mice which had received VV-priming and subsequent VV-injection into the tumor mass exhibited complete tumor regression. On the contrary, mice which had received mere intratumoral VV-injection in the absence of VV-priming failed to exhibit appreciable tumor regression. Mice (regressor mice) whose tumor had completely regressed by the above VV-immunotherapy were shown to have acquired systemic anti-tumor immunity which was confirmed by a challenge with syngeneic tumor cells after the tumor regression. In vitro analysis of these immune mice revealed that potent tumor-specific cytotoxic T lymphocyte (CTL) responses were preferentially induced, but with no detectable anti-tumor antibody responses. Such a potent tumor specific immunity was not observed in mice which had received intratumoral VV-injection in the absence of VV-priming.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Reticuloendotheliosis virus-transformed cells contain infectious and noninfectious proviral sequences in different chromosomes.
The integration site of reticuloendotheliosis virus proviral DNA in the DNA from a cloned reticuloendotheliosis virus-transformed bone marrow cell line was studied. These reticuloendotheliosis virus-transformed bone marrow cells produce a replication-defective transforming virus (REV-T) and a nontransforming helper virus, designated reticuloendotheliosis-associated virus (REV-At). The DNA from this REV-T transformed bone marrow cell line was hybridized with 3H-labeled in vitro synthesized DNA complementary to the reticuloendotheliosis virus (RE) genome to show that these cells contain approximately five genome equivalents per haploid genome. These hybridization experiments did not distinguish between the transforming and nontransforming virus sequences in these cells. Metaphase chromosomes have been isolated from colcemid-treated bone marrow cells and separated into four different size classes on zonal gradients. Hybridization experiment with RE-specific cDNA indicated that all four size classes of chromosomes contain RE-specific sequences. To locate the infectious provirus DNA of the helper virus REV-At, transfection experiments were performed with DNA extracted from the chromosomes of each size class. Infectious REV-At provirus DNA was found principally in one size class. This size class contained the intermediate size macrochromosomes 5 through 11. These results suggest that REV-At proviral DNA must be integrated into a specific chromosome to produce infectious virus.
Animals↗