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PubMed1994

Human herpesvirus 6 (HHV-6) is a helper virus for adeno-associated virus type 2 (AAV-2) and the AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA replication and regulate gene expression.

We have previously described the apparent acquisition by human herpesvirus 6 (HHV-6) of the multifunctional rep gene of the helper-dependent human parvovirus adeno-associated virus type 2 (AAV-2). We report here that HHV-6 is a full helper virus for AAV-2 replication, suggesting a mechanism for transfer of the rep gene between the two viruses by recombination of replicative intermediates. The HHV-6 rep gene cloned under control of the human cytomegalovirus immediate early promoter complemented replication of a rep-deficient AAV-2 genome. In cotransfection experiments with heterologous promoters linked to the CAT reporter gene, HHV-6 rep activated the human immunodeficiency virus (HIV) long terminal repeat (LTR) in fibroblast cell lines but not in T-cells. In contrast, AAV-2 rep inhibited HIV LTR activity in both fibroblast and T-cell lines. The effect of HHV-6 and AAV-2 rep genes on the HIV LTR was independent of the NF-kappa B, Sp1, and TATA box elements. These results suggest that HHV-6 Rep is a multifunctional regulatory protein with properties related to, but distinct from, those of AAV-2 Rep.

Cell Line↗
PubMed2005

Soybean mosaic virus helper component-protease enhances somatic embryo production and stabilizes transgene expression in soybean.

Soybean mosaic virus (SMV) helper component protease (HC-Pro), a suppressor of post-transcriptional gene silencing, was evaluated for its ability to enhance production of soybean hygromycin-resistant somatic embryos (HR-SEs), and stabilize transgene expression. Immature soybean cotyledonary explants were co-cultured with Agrobacterium tumefaciens strain KYRT1 harboring either pCAMBIA1302, carrying a hygromycin phosphotransferase gene (hpt) and a gene encoding green fluorescent protein; pCAMBIA1305.1, carrying hpt and beta-glucuronidase (uidA) genes; pG2-HC-Pro, a derivative of pCAMBIA1305.1 containing SMV G2 HC-Pro; or pG5-HC-Pro, a derivative of pCAMBIA1305.1 containing SMV G5 HC-Pro, but lacking uidA. Significantly (rho<0.02) higher numbers of HR-SEs were obtained from explants transformed with Agrobacterium harboring either pG2-HC-Pro or pG5-HC-Pro than with either of the vector controls (pCAMBIA1302 or pCAMBIA1305.1). Beta-glucuronidase (GUS) expression was significantly (rho<0.003) higher in 50-day-old transgenic plants expressing GUS along with SMV-HC-Pro and in SMV-infected GUS transgenic plants than in transgenic plants expressing GUS alone. Together, these data suggest that SMV-HC-Pro enhanced recovery of HR-SEs by suppressing silencing of the hygromycin phosphotransferase gene.

Base Sequence↗
PubMed1977

Effect of helper virus on the number of murine sarcoma virus DNA copies in infected mammalian cells.

Cell lines of four mammalian species were each examined for the number of Moloney murine sarcoma virus (M-MSV) DNA copies in total cellular DNA after M-MSV transformation. Sarcoma-positive, leukemia-negative (S+L-) M-MSV-transformed cells were compared to M-MSV-transformed cells infected with a replicating leukemia virus. Both unfractionated M-MSV complementary DNA and complementary DNA representing the MSV-specific and the MSV-murine leukemia virus-common regions of the M-MSV genome were hybridized to total cellular DNA of various species. DNAs of mouse, cat, dog, and human S+L-cells contained from less than one to a few proviral M-MSV DNA copies per haploid genome. In contrast, helper virus-coinfected, M-MSV-producing cells of each species showed a 3- to 10-fold increase in M-MSV proviral DNA over that found in corresponding S+L- cells. MSV-specific and MSV-murine leukemia virus-common nucleotide sequences were each increased to a similar degree. A corresponding examination of cellular DNA of leukemia virus-infected normal or S+L- mammalian cells was performed to establish the resulting number of leukemia proviral DNA copies. The infection of normal or S+L- mammalian cells with several leukemia-type viruses that did not have nucleotide sequences closely related to the cell before infection resulted in the appearance of one to three corresponding leukemia proviral DNA copies.

Animals↗
PubMed1993

Mutations in zucchini yellow mosaic virus helper component protein associated with loss of aphid transmissibility.

Zucchini yellow mosaic virus (ZYMV) is a potyvirus transmitted by aphids in a non-persistent manner. Isolates having partially or totally lost their ability to be transmitted by aphids have been isolated and found to be affected in their helper component activities. We have sequenced the helper component coding region of poorly aphid-transmissible (PAT) variants of two strains of ZYMV, E15 and R5A. Mutations have been identified at the nucleotide level leading to two amino acid changes in the E15 PAT variant helper component and to one amino acid change located in the cysteine-rich region (well-conserved among potyviruses) in R5A PAT variant helper component. The mutation in the R5A variant changes the same amino acid as the one identified in potato virus C, a non-transmissible strain of potato virus Y.

Amino Acid Sequence↗
PubMed1979

Defective retrovirus-like 30S RNA species of rat and mouse cells are infectious if packaged by type C helper virus.

RNA species with properties of defective retrovirus-like 30S RNA genomes have previously been detected in both rats and mice and in some rat and mouse retroviruses. Using cell lines which express high levels of this retrovirus-like RNA, we formed pseudotypes of the 30S RNAs with helper-independent type C viruses. A pseudotype virus complex containing a mouse 30S subunit was transmitted to rat cells, and a pseudotype virus complex containing a rat 30S subunit was transmitted to bat cells. In other transmission experiments, a rat 30S subunit was isolated in nonproducer bat cells without detectable expression of the helper-independent type C virus used to pseudotype it. The results provide further support for the retrovirus-like nature of the rat 30S subunit and provide evidence which supports the protovirus hypothesis proposed by Temin.

Animals↗
PubMed1994

Mapping helper virus functions for cucumber mosaic virus satellite RNA with pseudorecombinants derived from cucumber mosaic and tomato aspermy viruses.

P-TAV is a strain of tomato aspermy virus (TAV) able to efficiently support the systemic accumulation of some (i.e., B2-satRNA) but not of other (i.e., Ix-satRNA) strains of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) in both tobacco and in tomato. As reported for V-TAV, the failure to support the systemic accumulation of Ix-satRNA seems to be due to an inefficient support of its systemic movement. Pseudorecombinants obtained by the exchange of RNAs 1 + 2 between P-TAV and Trk7-CMV, an efficient helper for the systemic accumulation of Ix-satRNA, were assayed for their ability to support the accumulation of CMV-satRNAs in tobacco plants and protoplasts. Pseudorecombinants having RNAs 1 + 2 from CMV supported the systemic movement and accumulation of CMV-satRNA as efficiently as CMV, whereas pseudorecombinants having RNAs 1 + 2 from TAV supported the CMV-satRNA very poorly. Thus, the ability to support the systemic movement and accumulation of CMV-satRNA is determined primarily by RNAs 1 + 2 and not by RNA 3, which is presumed to encode movement functions in the cucumoviruses and only has a minor, modulating effect on the systemic accumulation of satRNA. This suggests that for systemic movement CMV-satRNA has to interact with (the gene products of) RNAs 1 and/or 2 or that these viral RNAs compete with the satRNA for interaction with the coat or other movement proteins.

Cucumovirus↗
PubMed1992

Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.

Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.

Animals↗
PubMed2000

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV.

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 +/- 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor alpha (51 +/- 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38- subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 +/- 7% of CD34+ progenitors and 21 +/- 6% of CD34+, CD38- progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient. Gene Therapy (2000) 7, 183-195.

ADP-ribosyl Cyclase↗
PubMed1993

Immunodetection of the plum pox virus helper component in infected plants and expression of its gene in transgenic plants.

Tobacco plants (Nicotiana tabacum cv. Xanthi) have been transformed via Agrobacterium tumefaciens vectors, with cDNAs corresponding to the plum pox virus (PPV) cistron 2 encoding helper component (HC-Pro) and with the first two and half cistrons of the PPV genome. Presence of the HC-Pro in PPV-infected plants and transgenic plants transformed with the gene coding for this protein was investigated using specific polyclonal antibodies produced against the PPV HC-Pro. The results suggest that two proteases are involved in the processing of the PPV N-terminal polyprotein to yield a protein of 48 k (HC-Pro). HC-Pro autolytically cleaves at its carboxyl-terminus and a proteolytic activity, probably associated with the protein (P1) encoded by the cistron 1, is required for the cleavage in planta between the proteins derived from cistrons 1 and 2.

Agrobacterium tumefaciens↗
PubMed1982

Adeno-associated virus helper activity of adenovirus DNA binding protein.

The requirement for the adenovirus (Ad) single-stranded DNA binding protein (DBP) in the expression of adeno-associated virus (AAV) proteins was studied by specific immunofluorescent staining of infected cells and in vitro translation of RNA from infected cells. The Ad5 mutant ts125, which carries a mutation in the DBP gene, helped AAV as efficiently as the Ad5 wild type (WT) did at both the permissive (32 degrees C) and nonpermissive (40.5 degrees C) temperatures in HeLa and KB cells. Furthermore, at 40.5 degrees C ts125 was as efficient as Ad5WT was in inducing the expression of AAV proteins in a line of Detroit 6 cells which is latently infected with AAV. However, little if any AAV protein was synthesized when coinfections were carried out with Ad5WT in CV-C cells, a monkey cell line that is highly restrictive for human Ad replication unless the cells are also infected with simian virus 40. On the other hand, AAV protein was efficiently produced in CV-C cells in coinfections with the Ad5 mutant hr404, whose growth is unrestricted in CV-C cells and whose mutation also maps in the DBP gene. Finally, preparations of cytoplasmic RNA extracted from CV-C cells infected with AAV and Ad5WT or from CV-C cells infected with AAV, Ad5WT, and simian virus 40 were each capable of directing the in vitro synthesis of abundant amounts of AAV proteins in a rabbit reticulocyte lysate system. These results indicate that the abnormal DBP of ts125 still retains its helper function for AAV replication, but that the molecular feature of the DBP which relates to the monkey cell host range restriction of Ad's may also account for the observed block to AAV protein translation in CV-C cells.

Adenoviruses, Human↗
PubMed1986

Functional analysis of the adenovirus type 5 DNA-binding protein: site-directed mutants which are defective for adeno-associated virus helper activity.

We generated four point mutations in the DNA-binding protein (DBP) gene of adenovirus type 5 by oligonucleotide-directed site-specific mutagenesis. The sites mutated were in the three conserved regions (CR; amino acids 178-186 [CR1], 322-330 [CR2], and 464-475 [CR3]) identified previously by comparative sequence analysis (G. R. Kitchingman, Virology 146:90-101, 1985). The mutations resulted in changes in amino acids 181 (Trp to Leu), 323 (Arg to Leu), 324 (Trp to Leu), and 469 (Phe to Ile). The mutated DBP genes were put under the control of the simian virus 40 early promoter and analyzed by transfection for their ability to help adeno-associated virus replicate its DNA in COS-1 monkey cells. Mutations in the aromatic amino acids 324 and 469 reduced the amount of AAV DNA replication approximately 10-fold, while the mutation in Arg 323 produced a reduction of approximately fourfold. The Trp-to-Leu mutation in amino acid 181 had no effect on AAV DNA replication. The decreased helper activity of the 323, 324, and 469 mutations was not caused by any effect of the mutation on the stability of the DBP. These results suggest that CR2 and CR3 are involved in AAV helper activity, specifically in AAV DNA replication. The relevance of these findings to the identification of residues important for the functions of DBP in adenovirus infection is discussed.

Adenoviruses, Human↗