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At least 325 records · Page 18Linked to original sources
PubMed1995

Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA.

We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid. These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus. One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame. When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus. Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus. The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes. These findings identify 41% of EBV DNA which is sufficient to immortalize primary human B lymphocytes and provide an assay to distinguish virus contributions to initiation or maintenance of cell proliferation or both. They also identify EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation.

Amino Acid Sequence↗
PubMed1988

Establishment of tumor-specific immunotherapy model utilizing vaccinia virus-reactive helper T cell activity.

It was previously demonstrated that preimmunization of mice with live vaccinia virus (VV) and subsequent immunization with VV-infected (modified), syngeneic tumor cells resulted in enhanced induction of tumor-specific immunity through a cellular collaboration between VV-reactive helper T (VV-Th) cells and tumor-specific effector cell precursors. On the basis of this augmenting system, a tumor-specific immunotherapy model was established in which a growing tumor regressed. C3H/HeN mice were pretreated with cyclophosphamide to eliminate nonspecific suppressor T cell activity and subsequently inoculated (primed) s.c. with live VV, leading to augmented induction of VV-Th cell activities. Four weeks later, the mice were inoculated i.d. with syngeneic X5563 myeloma cells. Six days after the tumor cell inoculation, live VV was injected into the tumor mass three times at 2-day intervals. Seven of ten 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 whose tumor had completely regressed (regressor mice) 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 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 an intratumoral VV injection in the absence of VV priming. Thus the results clearly indicate that the tumor regression was accompanied by concurrent generation of a potent tumor-specific immunity, suggesting that the cellular collaboration between VV-Th cells and tumor-specific effector cell precursors is also functioning in this VV-immunotherapy protocol, likewise the immunoprophylactic model. Therefore, the present model provides an effective maneuver for tumor-specific immunotherapy and this system will be theoretically applicable to human cancer treatment.

Animals↗
PubMed1992

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.

Animals↗
PubMed1998

Encapsidation studies of poliovirus subgenomic replicons.

The inclusion of a foreign marker gene, chloramphenicol acetyltransferase (CAT) gene, into the poliovirus genome allows its replication and encapsidation to be easily monitored using a simple enzyme assay. Such poliovirus replicons require the presence of helper virus for their successful propagation and thus are similar to defective interfering (DI) viruses. In genomes containing the CAT gene, the majority of the P1 virus capsid region of the poliovirus genome could be removed without destroying viability. The smallest replicon was significantly smaller than any naturally occurring DI particle so far reported, yet it retained the ability to replicate and be encapsidated by structural proteins provided by helper virus in trans. The efficiency with which the replicons were encapsidated was investigated using a direct immunostaining technique that allows individual cells infected with either a replicon or helper virus to be quantified. These results were compared to the frequencies of trans-encapsidation of polioviruses and coxsackievirus B4 using a two-stage neutralization assay. Poliovirus types 1, 2 and 3 but not coxsackievirus B4, coxsackievirus A21 or rhinovirus 14 provided efficient trans-encapsidation of poliovirus type 3 or type 3-derived replicons. These results suggest that a specific encapsidation process operates and that it does not involve RNA sequences within the region of the genome encoding the capsid proteins.

Animals↗
PubMed1998

In situ cancer vaccination: an IL-12 defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity.

Intratumoral inoculation of replication-competent, attenuated herpes simplex virus (HSV) mutants inhibits tumor growth by direct cytotoxic viral replication and induction of a tumor-specific immune response. To boost the antitumor response, we describe a defective HSV vector encoding IL-12 as an adjuvant to in situ vaccination by the replication-competent HSV helper virus. The defective HSV vector system consists of defective particles containing tandem repeats of the cytokine genes (p40 and p35) in combination with a HSV helper virus. Heterodimeric IL-12 was expressed and secreted after IL-12 defective vector infection of tumor cells. In a syngeneic, bilateral established tumor model with CT26 murine colon carcinoma, unilateral intratumoral inoculation with an IL-12 defective/replication-competent HSV vector combination significantly reduced tumor growth of the inoculated and noninoculated contralateral tumors. This antitumor effect was significantly greater than with a lacZ-defective/replication-competent HSV vector combination, which itself was significantly greater than the mock inoculation. Efficacy is associated with enhancement of tumor-specific CTL activity, including specificity against the CT26 immunodominant MHC class I restricted Ag AH1, and IFN-gamma production. There was no significant tumor growth inhibition after intratumoral inoculation of s.c. CT26 tumors in athymic mice. We conclude that this defective HSV vector system is an effective method for cytokine gene delivery to tumors in situ and IL-12 expression in tumors synergizes the antitumor activity mediated by the replication-competent HSV helper virus.

Animals↗
PubMed1979

Persistence and pathogenicity of defective Friend spleen focus-forming virus. Decreased transplantability of hemopoietic cells as a marker for preleukemic change.

A latent form of persistent infection can be established in susceptible adult mice inoculated with a preparation of defective Friend spleen focus-forming virus (SFFV) purified free from standard leukemia-inducing helper virus (LLV-F). SFFV persistence was initially observed using an in vivo rescue technique in which SFFV could be directly rescued to form splenic foci of malignant erythropoiesis in mice. At approximately 30 d after virus inoculation however, SFFV could not be rescued after inoculation of LLV-F indicating that persistently infected (i.e., SFFV+) mice were either immume to exogenous helper virus or able to express SFFV-associated defective-interfering (DI) function(s). Persistent infection by SFFV was further documented using an in vitro rescue technique and ultimately resulted in the induction by SFFV of erythroleukemia in the absence of polycythemia or overt virus production. However, SFFV rescued by LLV-F from persistently infected normal and transformed hemopoietic cells was able to induce polycythemia in adult mice suggesting that this is a helper controlled property of the Friend virus complex. Transplantable SFFV-induced erythroleukemic cells could be retrieved from persistently infected yet histologically normal mice. The duration of SFFV persistence in normal spleen tissue suggests that the SFFV provirus resides in either a long-lived or pluripotent hemopoietic cell. Further, certain changes occurred, presumably in the membranes of persistently infected cells, which preceded the overt development of Friend leukemia and facilitated the definition of an SFFV preleukemic phase. Cell surface alterations were revealed using cell transfer techniques. Hemopoietic cells harboring a rescuable SFFV failed to proliferate when inoculated into lethally irradiated, syngeneic adult mice. In contrast, the transformed progeny of preleukemic cell populations and spleen cells transformed by FV complex (i.e., cells replicating both SFFV and LLV-F) were not rejected. This result suggests that histologically normal SFFV+ preleukemic cells express an antigen recognition site which is not present on overtly transformed cells and which may be a pertinent surveillance target for host anti-leukemogenic reactions.

Animals↗
PubMed1979

Persistence of infectious Friend virus in spleens of mice after spontaneous recovery from virus-induced erythroleukemia.

Persistent infectious virus was detected in the majority of spleens of (C57BL/10 X A.BY)F1 mice after spontaneous recovery from Friend virus-induced erythroleukeima. The Friend murine leukemia helper virus (F-MuLV) was detected in titers up to 3 X 10(5) PFU/g of spleen. The defective spleen focus-forming virus (SFFV) was present in much lower titers and could be detected in cell-free spleen homogenates only after amplification of virus titer by growth of virus in vitro on SC1 cells. The incidence of cells producing F-MuLV alone in spleens after recovery from leukemia was 0.003 to 0.3%, and the incidence of cells producing both F-MuLV and SFFV was less than 0.0001 to 0.01%. In most recovered mouse spleens there appeared to be a selective reduction of SFFV relative to F-MuLV.

Animals↗
PubMed1991

A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication.

Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.

Chromosome Mapping↗
PubMed1992

Transmission of dengue virus-induced helper signal to B cell via macrophages.

The helper T cells (TH) generated in dengue type 2 virus (DV) infection of mice produce a soluble helper cytokine (HF) which enhances the clonal expansion of DV-specific IgM antibody plaque forming cells (PFC). The present study was undertaken to investigate the mechanism of transmission of the helper signal from TH and HF to B cells. It was observed that TH could transmit the helper signal to B cells by direct cell to cell contact, but HF could not do so without the presence of live macrophages (M phi). HF was adsorbed by both heat killed and live M phi but the former could not transmit it to B cells. Both the polypeptide chains of HF bind to M phi. HF remains on the surface of M phi and can be retrieved completely by contact with B cells for 40 min. The helper signal from TH or HF-adsorbed M phi could not be transmitted to B cells when they were separated from each other by a cell impermeable membrane. The enhancement of PFC count is greater when the signal is transmitted by HF-adsorbed M phi as compared to that by TH alone. Thus, even with lower frequency of TH a significant number of B cells may be triggered with the help of HF and M phi. The findings thus show that the DV-specific helper signal could be transmitted only by a close physical contact of the plasma membranes of the signal presenting cells (TH or HF-adsorbed M phi) and B cells.

Animals↗
PubMed1988

Construction of a safe and efficient retrovirus packaging cell line.

Ecotropic and amphotropic retrovirus packaging cell lines have been constructed in which the helper virus genome have been separated onto two plasmids, and the psi packaging signal and 3' LTR have been removed. The gag and pol genes on one plasmid and the env gene on another plasmid were transfected into NIH 3T3 cells. Packaging cell lines produced by these transfected genes released titers of replication-defective retroviral vectors which were comparable to titers produced by packaging cell lines containing the helper virus genome on one plasmid. There has been no evidence of recombination events between the ecotropic helper virus plasmids and the vector virus plasmid that would result in the generation of intact replication-competent virus. These results suggest that a packaging cell line containing gag, pol and env on different plasmids is efficient and safe for use in retroviral gene gransfer.

Cell Line↗
PubMed1991

Dengue virus-induced helper cytokine has two polypeptide chains which bear different determinants.

Dengue type 2 virus (DV) induces generation of a T cell helper cytokine (HF) in mouse spleen which enhances the antigen-specific antibody plaque forming cell count in syngeneic mice. The present study was undertaken to investigate the molecular structure of HF. It was observed that the activity of HF was abrogated by treatment with reducing agents such as glutathione, ouabain or dithiothreitol (DTT) which cleave disulphide bonds, separating the polypeptide chains. The two polypeptide chains could be purified by high performance liquid chromatography of DTT treated HF. The individual chains had no helper activity, but it could be restored by mixing the two. One chain of the HF bonded to the DV-antigen coupled immunosorbent column and the other to the anti-I-Ak antibody coupled column. Thus, DV-induced HF is a disulphide bonded double chain structure, one chain having antigen and the other having I-A determinants; the presence of both chains is essential for helper activity.

Animals↗
PubMed1971

Chemical induction of focus-forming virus from nonproducer cells transformed by murine sarcoma virus.

Focus-forming virus can be induced by chemicals from virus-negative clonal lines of cells transformed by murine sarcoma virus. The viruses activated from such nonproducer cells have the host range and serologic properties of endogenous helper viruses of the cells rather than those of the sarcoma virus used to transform them. The evidence indicates that murine sarcoma virus-transformed nonproducer cells of two species contain the genetic information for both murine sarcoma and helper virus production in an unexpressed form.

Amines↗
PubMed2000

Fifty-one kilobase HSV-1 plasmid vector can be packaged using a helper virus-free system and supports expression in the rat brain.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have a number of attractive features for gene transfer into neurons. In particular, the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to accommodate large inserts. We now report the construction and characterization of a 51 kb HSV-1 plasmid vector. This vector was efficiently packaged into HSV-1 particles using a helper virus-free packaging system. The structure of the packaged vector DNA was verified by both Southern blot and PCR analyses. A vector stock was microinjected into the rat striatum, the rats were sacrificed at 4 days after gene transfer, and numerous X-gal positive striatal cells were observed. This 51 kb vector was constructed using general principles that may support the routine construction of large vectors. Potential applications of such HSV-1 vectors include characterizing large promoter fragments or genomic clones and co-expressing multiple genes.

Animals↗
PubMed2005

Novel strategy for generation and titration of recombinant adeno-associated virus vectors.

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

Animals↗
PubMed1995

A conserved motif at the 3' end of mouse hepatitis virus genomic RNA required for host protein binding and viral RNA replication.

A conserved 11-nucleotide sequence, UGAAUGAAGUU, at the 3' end of the genomic RNA of coronavirus mouse hepatitis virus was required for host protein binding and viral RNA synthesis. An RNA probe containing this 11-nucleotide sequence bound four cellular proteins with a highly labeled protein of 120 kDa and three minor species with sizes of 103, 81, and 55 kDa. Mutation of the 11-nucleotide motif abolished cellular protein binding. The RNA-protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable from those observed with extracts from uninfected cells. Both negative-strand synthesis and positive-strand replication of viral defective interfering RNAs in the presence of helper virus were affected by mutations that disrupt RNA-protein complex formation, even though the 11 mutated nucleotides were converted to the wild-type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred relatively early in the MHV replicative cycle at 5.5 to 7.5 hr postinfection, a time when viral RNA synthesis and replication of positive-strand DI RNA were at barely detectable levels. A DI RNA with a mutation upstream of the protein binding element replicated as efficiently as wild type without undergoing recombination. Thus, the 11-nucleotide conserved host protein binding motif appears to play an important role in viral RNA replication.

Animals↗
PubMed1983

Characterization of the early and late stages of myelomonocytic leukemia induced by Friend helper-independent virus F-MuLV: isolation and induction of Friend myelomonocytic leukemic cell lines.

With the use of cloned helper-independent Friend leukemia virus (F-MuLV), we have induced a high incidence (approximately 70%) of myelomonocytic leukemia in mice resistant (Fv-6rr or Fv-6rs) to erythroleukemia induction by this virus. The spleen cells from these mice (DBA/2 or BALB/c X DBA/2) were found to contain a high level of progenitor cells capable of forming granulocytic and macrophage colonies (CFU-GM). These CFU-GM, however, were different from those in the spleens of uninfected mice, as they were either very sensitive to or independent of conditioned medium. No erythroid progenitor bursts (BFU-E) or precursor (CFU-E) cells were detected in the spleens of these diseased animals. If these mice with myelomonocytic leukemia were kept alive by transfusion of red blood cells from uninfected mice, tumorigenic cell lines, capable of being transplanted, into adult mice can be isolated. Three such cell lines TTA-1, TTA-3, and TTA-9 have been established, and they retain their morphology of monocytes and macrophages as well as being positive for the monocyte-specific stain alpha-naphthyl-acetate esterase. These myelomonocytic leukemia cell lines can also be induced in culture by spleen cell-conditioned medium to differentiate into macrophages. Other conditioned media such as L-cell-conditioned medium, phytohemagglutinin-stimulated leukocyte-conditioned medium, and WEHI-3 conditioned medium were less effective in their abilities to stimulate differentiation in these myelomonocytic leukemia cell lines.

Animals↗
PubMed1997

Assembled coronavirus from complementation of two defective interfering RNAs.

In the presence of an RNA- temperature-sensitive (ts) mutant helper virus, two coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNAs complemented each other, resulting in the assembly of MHV particles; we used this ability to complement as a means to study coronavirus assembly. One of the two DI RNAs was DIssA, a naturally occurring self-replicating DI RNA encoding N protein and the gene 1 proteins that encode RNA polymerase function; DIssA supports the replication and transcription of other non-self-replicating DI RNAs. The other DI was a genetically engineered DI RNA that encoded sM and M proteins. Coinfection of these two DIs at the nonpermissive temperature for the ts helper virus resulted in replication and transcription of both DI RNAs but not in synthesis of the helper virus RNAs. MHV particles containing DI RNAs, N protein, and M protein, all of which were exclusively derived from the two DI RNAs, were released from the coinfected cells; the amount of sM protein was below the limits of detection. Analyses of DI RNAs with mutations in the two envelope protein genes demonstrated that M and sM proteins appeared to be required for assembly and release of MHV particles that contained DI RNAs and N protein, while S protein was not required for assembly and release of MHV particles.

Animals↗
PubMed2001

Infection of purified nuclei by adeno-associated virus 2.

Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, requires co-infection with a helper virus for its optimal replication. Although AAV possesses a broad host range, certain cell types lack the machinery necessary for efficient entry into the cell and intracellular trafficking of AAV into the nucleus, where the viral second-strand DNA synthesis must occur before gene expression. We have demonstrated that in less-permissive mouse fibroblasts, the virus fails to transport to the nucleus due to altered endocytic processing. However, relatively little is known about the intracellular site of viral uncoating and transport of the virion across the nuclear envelope. Here, we provide evidence that AAV can efficiently enter intact nuclei purified from both permissive and less-permissive cell types. Furthermore, entry into the nucleus is time- and temperature-dependent, but is not saturable and seems to occur independently of the nuclear pore complex. We also demonstrate that purified nuclei contain all of the machinery necessary for uncoating and viral second-strand DNA synthesis even in the absence of a helper virus. These studies provide new insights into the basic biology of AAV and may also have implications for the optimal use of AAV vectors in human gene therapy.

3T3 Cells↗