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PubMed1998

Rescue of IBV D-RNA by heterologous helper virus strains.

Coronavirus defective RNA (D-RNA) vectors could be developed to deliver selected genes for the production of recombinant coronavirus vaccines. An IBV D-RNA, CD-61, derived from a naturally occurring IBV Beaudette D-RNA, CD-91, is being developed as a D-RNA vector for IBV. In order to use CD-61 as a vector it will require rescue by heterologous strains in addition to Beaudette. Rescue will be determined by recognition of replication and packaging signals within the D-RNA by the helper virus. The 5' and 3' UTRs are believed to contain sequences involved in replication and transcription. The 5' and 3' UTRs of six strains of IBV have been sequenced and experiments performed using six strains of helper virus for rescue of CD-61 to determine whether rescue correlates with sequence conservation within the 5' and 3' UTRs. Results indicate that all strains of helper virus rescued the D-RNA to varying degrees. Sequence comparisons show a high degree of sequence identity in the UTRs, but enough strain differences exist to be used as markers. The 5' and 3' UTRs of the D-RNAs rescued by the heterologous strains were also sequenced and leader switching between the helper virus and the Beaudette leader on the D-RNAs was observed.

3' Untranslated Regions↗
PubMed1995

Differential effects of satellite RNA on the accumulation of cucumber mosaic virus RNAs and their encoded proteins in tobacco vs zucchini squash with two strains of CMV helper virus.

The presence of cucumber mosaic virus (CMV) satellite RNA usually reduces the yield of accumulated helper virus, although more so in solanaceous than in cucurbit hosts. The accumulation of viral RNA and viral-encoded proteins of two strains of CMV (Fny- and Sny-) known to differ in their ability to support satellite RNA in zucchini squash was examined in squash and tobacco to determine the effect of satellite RNA on the accumulation of viral-associated components. In the absence of satellite RNA, Fny- and Sny-CMV showed similar levels of accumulation of RNA at 7 days postinoculation (p.i.), but by 14 days p.i. the Fny-CMV RNAs accumulated to lower levels than did both strains at 7 days p.i., in either host. The levels of accumulated Sny-CMV-encoded proteins were higher than those encoded by Fny-CMV in tobacco, but not squash plants, at 7 days p.i. At 14 days p.i., for Fny-CMV vs Sny-CMV, there were differences in the levels of accumulation of most CMV-encoded proteins in both hosts, more exacerbated in tobacco vs squash. The effect of satellite RNA was to intensify these differences; that is, by 7 days p.i., satellite RNA reduced the accumulation of Fny-CMV RNAs 1 and 2 and their encoded proteins in both tobacco and squash but had little or no effect on the accumulation of Sny-CMV RNAs or encoded proteins. By 14 days p.i., the levels of accumulation of all Fny-CMV RNAs and encoded proteins were severely reduced in both hosts, and the levels of accumulation of Sny-CMV RNAs 1 and 2 and their encoded proteins were also reduced in tobacco, but not squash. Sny-CMV did not support satellite RNA accumulation in squash plants or protoplasts. Satellite RNA did not appear to have a direct effect on the movement of either CMV strain. Rather, accumulation studies in tobacco protoplasts indicated that the difference in response of Fny-CMV vs Sny-CMV to satellite RNA in tobacco was due to the extent to which satellite RNA affected the levels of RNA 1, and to a lesser extent RNA 2, and their encoded proteins, 1a and 2a, both components of the CMV replicase.

Cucumber Mosaic Virus Satellite↗
PubMed1997

Support of a cucumber mosaic virus satellite RNA maps to a single amino acid proximal to the helicase domain of the helper virus.

Cucumber mosaic virus (CMV) is a tripartite RNA virus that can support the replication of satellite RNAs, small molecular parasites of the virus. Satellite RNAs can have a dramatic effect on the helper virus and the host plant in a manner specific to the helper, satellite, and host. Previously, we showed that the Sny-CMV strain is not able to support the replication of the WL1 satellite RNA in zucchini squash and that this phenotype maps to RNA 1. In the present study, we use recombinant cDNA clones of Fny- and Sny-CMV RNA 1 and a site-directed mutant of Fny-CMV RNA 1 to demonstrate that the inability to support WL1 satellite RNA maps to a single amino acid at residue 978 in the 1a protein, proximal to the helicase domain VI. Support of satellite RNA in whole plants and in protoplasts of zucchini squash is analyzed.

Amino Acid Sequence↗
PubMed1982

Molecular cloning of the endogenous rat C-type helper virus DNA sequence: structural organization and functional analysis of some restricted DNA fragments.

Recently, we have identified and purified the integrated and proviral DNA sequences specific for two endogenous rat type C leukaemia helper viruses: WR-RaLV which originated from a fibrosarcoma induced in a feral rat and RHHV from the cell line HTC-H1 which originated from a Buffalo rat hepatoma. The rat leukaemia helper virus DNA sequences have previously been shown to be 8.4 to 8.8 kilobases (kb) in size. In this communication, we report the molecular cloning of the 8.8 kb DNA of RHHV by ligation at the BamHI site of the vector pBR322, cultured in an Escherichia coli RR1 host. After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using 32P-labelled RHHV cDNA, four clones were isolated, two of which carried the total 8.8 kb DNA. A detailed restriction endonuclease map of the cloned RHHV DNA was deduced by sequential digestions of either 3'- or 5'-labelled DNA. Of the 14 restriction enzymes tested, EcoRI, BamHI, PstI, KpnI, TaqI, PvuII and SmaI gave informative cleavage patterns. At least two copies of long terminal repeated sequences (LTR) flanking the 3' and 5' termini of the proviral DNA were identified by TaqI and PstI cleavages. LTR in the rat endogenous leukaemia helper virus DNA measured 780 +/- 20 nucleotides in length. The genetic information encoded by the cloned DNA was also analysed by hybridization selection of RHHV mRNA, which was then used in cell-free protein synthesis in a rabbit reticulocyte lysate system. Essentially all major RaLV-specific proteins precipitable by anti-RaLV serum were synthesized in vitro, confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.

Animals↗
PubMed1995

Variation in the hypervariable region of cucumber mosaic virus satellite RNAs is affected by the helper virus and the initial sequence context.

The D satellite RNA (sat RNA) of cucumber mosaic virus (CMV) was previously shown to contain a region of hypervariability around nucleotide 230, in wild-type populations and in cDNA clones and progeny of one such clone (pDsat4) after passage with the subgroup I strain Fny-CMV. This hypervariable region (HVR) consists of a series of consecutive A and/or U residues. We found that variability is also generated in the HVR of transcript derived from pDsat4 after passage with the subgroup II strain LS-CMV and with tomato aspermy virus (TAV). However, the progeny differ with respect to the sequence of the HVR after passage with both LS-CMV and TAV. Another D-sat RNA cDNA clone that contains a C residue in the HVR, pDsat1, was previously shown not to develop variability in the HVR upon passage with Fny-CMV. However, when the C (position 231) was changed to an A residue, variability developed by the third passage with Fny-CMV. An additional cDNA clone derived from the B1-sat RNA, pBsat5, also contains a C residue in the region analogous to the D-sat RNA HVR and did not develop variability upon passage with either Fny- or LS-CMV. Changing this C to a U residue did not result in the development of hypervariability in the progeny of transcript from this mutant. Models to explain the generation of hypervariability are discussed.

Base Sequence↗
PubMed1986

Transmission of (LTR, v-src, LTR) without recombination with a helper virus.

We have analyzed transmission of (LTR, v-src, LTR) cryptic structure integrated in the H-19 mammalian tumor cell line. From this cell line different isolates of transforming virus were rescued in heterokaryons produced by fusion with chicken fibroblasts infected by replication-competent avian leukosis virus RAV-1. One of them (F6) was used for the transformation of avian cells in the absence of the helper virus. In four transformed cell lines studied, the (LTR, v-src, LTR) structure was again integrated at a unique position in the cell DNA of each line. This indicated that the (LTR, v-src, LTR) structure is transmitted by the helper virus without recombination. This point has been further supported by the finding that a src-containing species corresponding in size to the nonpolyadenylated src mRNA is present in the RNA isolated from the rescued F6 transforming virus which might serve as template for the synthesis of (LTR, v-src, LTR) structure by the reverse transcriptase provided by RAV-1.

Animals↗
PubMed1983

Effect of murine host genotype on MCF virus expression, latency, and leukemia cell type of leukemias induced by Friend murine leukemia helper virus.

Leukemias induced by neonatal inoculations of several mouse strains with different strains of Friend murine leukemia helper virus (F-MuLV) were followed for time of disease onset, cytochemical analysis of predominant cell types in leukemic organs, and expression of infectious mink cell focus-inducing (MCF) viruses detected by mink cell foci or MCF-specific monoclonal antibodies. Most BALB.B and IRW mice had a rapidly appearing, severe anemia and hepatosplenomegaly consisting of erythroid cells. MCF viruses were usually isolated from enlarged spleens of IRW mice. In contrast, C57BL/10 mice had a lower incidence of disease and much slower course. Splenomegaly and lymphadenopathy with mild anemia were seen, and the predominant cell types were either myeloid (chloroleukemia) or lymphoid. MCF viruses were never isolated from this mouse strain. (C57BL/10 X IRW)F1 mice were intermediate in latency, but all mice had disease by 8 months. Myeloid, lymphoid, and some mixed leukemias with an erythroid component were observed, but in no case did we see the severe anemia or pure erythroid involvement typical of IRW and BALB.B mice. MCF viruses were, however, isolated from 22% of these mice regardless of leukemia cell type. DBA/2 mice had a disease pattern similar to the (C57BL/10 X IRW)F1 mice, and MCF viruses were isolated from three of six mice tested. Inoculation of IRW mice with the low virulence B3 strain of F-MuLV produced disease with a longer latency than F-MuLV 57, but similar cell types were transformed by both viruses. In vitro cell lines were derived from 14 mice, and most were tumorigenic in vivo. Three lines released infectious MCF virus, and three others expressed MCF-specific cell surface antigens but did not release virus. Eight lines expressed no MCF infectious virus or viral antigens. Several lines released infectious xenotropic viruses and/or expressed xenotropic MuLV cell surface antigens recognized by monoclonal antibodies reactive with xenotropic viruses. The lack of MCF expression in many primary leukemic tissues as well as in in vitro derived leukemia cell lines of C57BL/10 and (B10 X IRW)F1 mice suggested that MCF virus generation and expression may not be required for leukemogenesis in some mouse strains or in some hemopoietic lineages.

Animals↗
PubMedDate not supplied

[The enhancement of the penetration of the human immunodeficiency virus into cells with the aid of a helper virus].

Penetration of human immunodeficiency virus (HIV) into the cells of lymphoblastoid T-cell line H9 was studied using 35S-methionine-labeled virus by demonstration of virus-specific proteins in the cytoplasm of the infected cells. Purification of the virus by ultracentrifugation through 30% glycerol was shown to lead to virus aggregation and its partial destruction manifested by the loss of gp120 protein, therefore unlabeled concentrated virus was used mainly with subsequent determination of virus-specific proteins by immune blotting. The addition of Sendai virus inactivated with UV rays to HIV increased the amount of HIV associated with cells as well as the amount of virus-specific proteins in the cytoplasm of the infected cells.

Cells, Cultured↗
PubMed2006

An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development.

Recovery of recombinant, negative-strand, nonsegmented RNA viruses from a genomic cDNA clone requires a rescue system that promotes de novo assembly of a functional ribonucleoprotein (RNP) complex in the cell cytoplasm. This is accomplished typically by cotransfecting permissive cells with multiple plasmids that encode the positive-sense genomic RNA, the nucleocapsid protein (N or NP), and the two subunits of the viral RNA-dependent RNA polymerase (L and P). The transfected plasmids are transcribed in the cell cytoplasm by phage T7 RNA polymerase (T7 RNAP), which usually is supplied by infection with a recombinant vaccinia virus or through use of a stable cell line that expresses the polymerase. Although both methods of providing T7 RNAP are effective neither is ideal for viral vaccine development for a number of reasons. Therefore, it was necessary to modify existing technology to make it possible to routinely rescue a variety of recombinant viruses when T7 RNAP was provided by a cotransfected expression plasmid. Development of a broadly applicable procedure required optimization of the helper-virus-free methodology, which resulted in several modifications that improved rescue efficiency such as inclusion of plasmids encoding viral glycoproteins and matrix protein, heat shock treatment, and use of electroporation. The combined effect of these enhancements produced several important benefits including: (1) a helper-virus-free methodology capable of rescuing a diverse variety of paramyxoviruses and recombinant vesicular stomatitis virus (rVSV); (2) methodology that functioned effectively when using Vero cells, a suitable substrate for vaccine production; and (3) a method that enabled rescue of highly attenuated recombinant viruses, which had proven refractory to rescue using published procedures.

Animals↗
PubMed1996

The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus.

We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3' end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.

Animals↗
PubMed1987

Gene expression in adeno-associated virus vectors: the effects of chimeric mRNA structure, helper virus, and adenovirus VA1 RNA.

We used a recombinant plasmid containing an adeno-associated virus (AAV) genome to construct several vectors which express the gene for chloramphenicol acetyltransferase (CAT). We transfected four different AAV-CAT vectors into human 293 (adenovirus-transformed) cells and analyzed CAT activity. We show that, for vectors using the AAV p40 and p19 promoter, the chimeric AAV-CAT transcripts began from the correct 5' position but the basal level of CAT expression depended in part on the structure of the transcript. We also examined the effects of coinfection of the cells with the helper adenovirus or cotransfection with a plasmid which expressed the adenovirus translational control RNA, VA1 RNA. Cotransfection with plasmids containing the gene for VA1 RNA resulted in elevated levels of CAT activity. VA1 RNA stimulated translation of the chimeric mRNA. However, in two cases, the VA1 RNA apparently decreased the level of mRNA. These results suggest that in addition to its function in translation, VA1 RNA acts at a second site to alter cytoplasmic accumulation of some mRNAs. Infection with adenovirus increased CAT activity several-fold by increasing the cytoplasmic levels of the chimeric AAV-CAT transcript. When the CAT gene is inserted down stream of the AAV intron, adenovirus and not VA1 RNA alone increased CAT activity by promoting accumulation of a spliced transcript.

Adenoviruses, Human↗
PubMed1993

Specificity of satellite activation by tobacco necrosis virus correlates with nucleic acid hybridization pattern between helper virus isolates.

Tobacco necrosis virus (TNV) comprises over 20 different isolates which are usually classified on the basis of serological cross-reactivity of their virus particles or specific activation of satellite virus strains (STNV-1, -2, and -C). We have studied the relationships between five TNV isolates, TNV-A, -G, -CN, -D, and -AC36 which exhibit considerable differences in symptom formation on Phaseolus vulgaris. It is shown that, like TNV-A, TNV-G and -CN support the multiplication of STNV-1 and -2. The ability to activate STNV-1 and -2 is not completely correlated with the virulence of the TNV isolates on Phaseolus as TNV-CN infects Phaseolus very inefficiently. The RNAs of all STNV-1 and -2 supporting TNV isolates were detectable by Northern blot analysis using RNA probes derived from TNV-A, whereas the RNAs of the STNV-C activating isolates (TNV-D and -AC36) were only detected with a TNV-D-derived RNA probe. This indicates that the classification of the TNV isolates on the basis of satellite activation is representative of the evolutionary relationships between the isolates.

Base Sequence↗
PubMedDate not supplied

[Helper viruses of adeno-associated virus type 4 replication].

In replication of adeno-associated virus type 4 (AAV-4) the helper function may be performed by a non-defective virus from the same group of parvoviruses (Kilham virus). The synthesis of AAV-4 antigen was observed in a pig embryo kidney cell line, SPEV, chronically infected with Kilham virus, strain RV-13, 45--52 passages. A one-day-old SPEV-Kilham culture was infected with AAV-4. The AAV-4 antigen was detected by immunofluorescence at 6, 8, 12, 18 hours, 2, 3, 4, and 5 days after inoculation. During the first 2--4 days after inoculation the AAV-4 antigen was found in the nucleus and perinuclear zone, later in the cytoplasm. A "new" helper virus for AAV-4 replication has been found: simiancytomegalovirus in human embryo fibroblast cell culture permissive for the helper virus. In the systems where AAV-4 replicates, its antigen can be detected in the nucleus and perinuclear zone by IF. AAV-4 did not replicate in a system insensitive to the helper virus or under non-permissive conditions: at the time, the AAV-4 antigen localized only in the cell cytoplasm was detected.

Animals↗
PubMed1980

Helper virus is not required for in vitro erythroid transformation of hematopoietic cells by Friend virus.

The Friend polycythemia virus complex (FVP), consisting of the replication-defective spleen focus-forming virus (SFFV) and a helper Friend murine leukemia virus (MuLV-F), produces erythroleukemia within 2-3 weeks in vivo. We have recently reported in vitro transformation of bone marrow cells by FVP, producing clusters of erythroid colonies (erythroid bursts) 4-6 days after infection. In contrast to uninfected bone marrow cells, FVP-treated cells proliferated and differentiated (synthesized hemoglobin) in the absence of added erythropoietin, the physiologic regulator of erythropoiesis. The relative roles of helper murine leukemia virus (MuLV) and SFFV in the in vitro erythroid transformation have now been examined. Pseudotype studies and the finding that cloned MuLV-F (free of SFFV) did not induce burst formation indicated that SFFV was essential for this in vitro effect of FVP. Because SFFV could not be obtained free of helper MuLV, we assessed the requirement of MuLV in the transformation by kinetic analyses of helper-deficient and helper-excess FVP preparations. Whereas helper-excess FVP gave single-hit kinetics both in vivo and in vitro, the helper-deficient FVP followed multiple-hit kinetics when titrated for spleen focus formation in vivo. Addition of MuLV-F to helper-deficient FVP prior to injection resulted in a marked enhancement of spleen focus formation and a conversion from multiple-hit to single-hit kinetics. In contrast, titration of this same preparation for erythroid burst transformation in vitro yielded single-hit kinetics, and the addition of helper MuLV-F had no effect. The time course of burst development was similar with or without added MuLV-F. Unlike burst transformation, SFFV production by these infected cultures followed multiple-hit kinetics. Addition of MuLV-F at the time of infection led to an enhancement of SFFV production and conversion of the titration curve from multiple-hit to single-hit. These data are consistent with the idea that SFFV is competent for erythroid transformation in vitro, but requires helper MuLV for its replication.

Animals↗
PubMed1986

Induction of the autonomous stage of transformation in erythroid cells infected with SFFV: helper virus is not required.

The erythroleukemia induced by the Friend spleen focus-forming virus (SFFV) in mice exemplifies a multistep oncogenic process. Its sequential steps include a rapid polyclonal hyperplastic stage and a more slowly developing malignant stage characterized by autonomous erythroid cells. We report here that the helper virus normally present in mice infected by SFFV is not required for development of the second stage of transformation. In this study, mice were infected with a polycythemia-inducing variant of SFFV which was prepared as a helper-free stock (L. Wolff and S. Ruscetti, 1985, Science 228, 1549). Highly malignant cells could be detected in helper-free SFFV-infected mice by their transplantability into the omentum of sublethally irradiated mice, and erythroleukemia cell lines, typical of previously isolated Friend murine erythroleukemia cell lines, could be established from diseased spleens. Like their helper virus-containing counterparts, the lines established with helper-free SFFV are inducible for hemoglobin synthesis with a variety of chemicals, but not erythropoietin, and express p53, a marker of malignant transformation. Although the cells expressed SFFV encoded proteins, none expressed gene products of replication competent murine leukemia viruses.

Animals↗
PubMed2003

High level of transgene expression in primary chronic lymphocytic leukemia cells using helper-virus-free recombinant Epstein-Barr virus vectors.

OBJECTIVE: Epstein-Barr virus (EBV)-based vectors have favorable features for gene transfer, including a high transduction efficiency especially for B cells, large packaging capacity up to 150 kb pairs, and ability to infect postmitotic cells. Recombinant EBV was explored for transduction of primary human B-cell chronic lymphocytic leukemia (CLL) cells. MATERIAL AND METHODS: EBV vectors deleted for all oncogenic sequences and encoding terminal repeats (TR) essential for encapsidation, the lytic origin of replication (oriLyt) for DNA amplification, and the enhanced green fluorescent protein (EGFP) were packaged using an optimized, helper-virus-free method. Infectious EBV virions encoding EGFP (EBV/EGFP) with an infectious titer up to 2 x 10(6) per milliliter were generated. Primary leukemic cells from 14 patients with CLL were successfully transduced with EBV/EGFP at a very low multiplicity of infection (< 1). RESULTS: Transgene expression was detected in up to 85% of cells 48 hours after infection. Transduction was specifically mediated by EBV vectors because gene transfer was inhibited by an antibody (72A1) directed against the viral envelope glycoprotein gp350/220. Furthermore, transduction of CLL cells with packaged EBV vectors coding for EGFP but deleted for TR sequences (TR-) did not result in EGFP expression compared to TR+ vector constructs (p = 0.009). CONCLUSION: Helper-virus-free EBV-based gene transfer vectors hold promise for development of genetic therapies for CLL patients.

Adult↗