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PubMed1991

The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity.

Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been generated in vitro using infectious cDNA clones and used to demonstrate that strong determinants of the attenuation and temperature-sensitive phenotypes of P2/Sabin reside in the 5' 492 nucleotides. In this region of the genome the viruses differ only at nucleotides 437 and 481. The ts phenotype associated with the 5' noncoding region is expressed at different temperatures in different cell lines, suggesting an involvement of cellular factors which may be species specific. Suppression of both the ts and attenuation phenotypes correlates with an A-G mutation at nucleotide 481, although other changes are also involved.

Animals↗

PubMed1991

Complementation and recombination between alfalfa mosaic virus RNA3 mutants in tobacco plants.

Deletions were made in an infectious cDNA clone of alfalfa mosaic virus (AIMV) RNA3 and the replication of RNA transcripts of these cDNAs was studied in tobacco plants transformed with AIMV replicase genes (P12 plants). Previously, we found that deletions in the P3 gene did not affect accumulation of RNA3 in P12 protoplasts whereas deletions in the coat protein (CP) gene reduced accumulation 100-fold (A. C. van der Kuyl, L. Neeleman, and J. F. Bol, 1991, Virology 183, 687-694). In P12 plants deletions in the P3 gene reduced accumulation by about 200-fold and accumulation of CP deletion mutants was not detectable. When P12 plants were inoculated with a mixture of P3- and CP-deletion mutants, both mutants replicated efficiently and various amounts of full-length RNA3 molecules were formed by recombination. The observation that some P3 and CP mutants did not recombine at a detectable level after several passages in P12 plants demonstrated that mutations in the AIMV P3 and CP genes can be complemented in trans.

Frameshift Mutation↗

PubMed1992

Two strains of SIVmac show differential transactivation mediated by sequences in the promoter.

Two infectious molecular clones of simian immunodeficiency virus, SIVmac251 and SIVmac239, have very different in vivo properties, SIVmac239 being much more pathogenic than SIVmac251. To assess whether the in vivo differences between the two viruses would be reflected in transcriptional rates in vitro, transcriptional activity in the presence of the transactivation protein tat was analyzed by transient transfection assays in HUT-78 and U937 cells. Whereas the two promoters had similar basal activities (Anderson and Clements, 1991, J. Virol. 65, 51-60) the promoter of SIVmac239 was transactivated to a greater extent. Removal of sequences 5' to -225 and 3' to +18 maintained the basal activity, yet made the promoter unresponsive to tat. Addition of bases +19 to +149 reconstituted transactivation and decreased basal activity. Analysis of deletion mutants with reconstituted transactivation response region determined that differences between the two strains were maintained even when only the proximal sequences, -225 to +18 of the U3 and R region were placed upstream of the TAR sequences. This region contains four nucleotide differences and the potential Sp-1-binding sites, where there are an additional 11 bases in SIVmac239 that create a third potential Sp-1 site, compared to only 2 in SIVmac251. Transactivation in this assay system was found to correlate better to RNA differences shortly after transfection (12 hr) than later (46 hr).

Base Sequence↗

PubMed1992

Characterization of the stage(s) in the virus replication cycle at which the host-cell specificity of the feline parvovirus subgroup is regulated in canine cells.

Feline panleukopenia virus (FPLV), mink enteritis virus (MEV), and canine parvovirus (CPV) are classified as a host-range variants. They show different host-range specificity in vivo and host-cell specificity in vitro. For instance, FPLV and MEV cannot grow or can grow only inefficiently in canine cell lines such as MDCK and the canine fibroma cell line A72. Here we have studied the mechanism(s) by which the different cell tropism is mediated in vitro. When FPLV or MEV was inoculated to A72 cells, viral DNA replicated slightly, few viral-antigen-positive cells were detected, and the culture fluid contained the threshold level of infectivity. On the other hand, when an infectious molecular clone of MEV (pMEV) was introduced into A72 cells, viral DNA replicated efficiently, and the culture fluid of pMEV-transfected cells contained much higher infectivities than that of MEV-infected cells. In spite of the restrictive growth in A72 cells, MEV could bind to A72 cells as efficiently as CPV. No detectable viral RNA was produced in MEV-infected A72 cells. In contrast, efficient viral transcription occurred in pMEV-transfected A72 cells. These results suggest that the restrictive infections of MEV and FPLV in A72 cells are not mediated by the attachment of the virus to the cells or by the events occurring after the viral transcription. It appears to be caused by the stage(s) in the virus replication cycle, which exists between a postadsorptional step required for virus penetration and the initiation of viral transcription.

Aleutian Mink Disease Virus↗

PubMed1992

Cell-free transmission of Vif mutants of HIV-1.

To determine the phenotype of human immunodeficiency virus type 1 (HIV-1) defective in the production of Vif, mutations were introduced into the vif gene of infectious molecular clones of the LAI, MAL, and ELI strains. Previous results had demonstrated that viruses derived from these wild type clones display different replicative capacities on peripheral blood mononuclear cells (PBMC) and different tropisms for CD4-positive cell lines. Using cell-free infection, Vif mutants of LAI, MAL, and ELI were found to have delayed kinetics and to produce less virus than their corresponding wild type viruses when propagated on most permissive cell lines. An additional mutation in the vpr gene in the LAI strain had no effect on this phenotype. However, on one T cell line, the H9 cell line, two Vif mutants of LAI and a Vif.Vpr double mutant were unable to replicate. Furthermore, Vif mutants from all three strains were only able to establish a productive infection on PBMC by coculture and not by cell-free infection. No defects in the processing of the virion proteins or the release of particles in the Vif mutants were detected.

Amino Acid Sequence↗

PubMed1991

Poliovirus antigen chimeras.

Poliovirus, the aetiological agent of paralytic poliomyelitis, is arguably the best characterized of all animal viruses. Using recombinant-DNA technology, this information, together with the availability of infectious cDNA clones of the notably safe and efficacious live attenuated Sabin 1 vaccine strains of poliovirus, has enabled the creation of hybrid viruses (chimeras) possessing novel antigenicity. The potential applications of these 'epitope-presentation systems' include their use as immunogens, as antigens for serodiagnosis, and as vaccines.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Alphavirus expression systems: applications to mosquito vector studies.

In this review, Steve Higgs, Ann Powers and Ken Olson describe how alphavirus expression systems, based on infectious cDNA clones of Sindbis virus, constitute a novel RNA virus delivery system suitable for the efficient expression of heterologous gene products or RNA sequences in mosquito cells or adult mosquitoes. The technique permits ready assessment of molecular genetic-based methods of intracellular interference to infection and provides a powerful new tool for molecular biological studies in arthropods.

Journal Article↗

PubMed1992

Molecular basis of virulence and growth of hepatitis A virus in cell culture.

The ability of engineering variants of hepatitis A virus (strain HM175) to replicate in cell culture or to cause disease in marmosets was evaluated. Virus variants were encoded by chimeric genomes constructed from infectious cDNA clones of two viruses (wild type and cell-culture-adapted) which differed in their ability to grow in vitro and to cause acute hepatitis in marmosets. Transfection and infectivity assays indicated that virus growth in vitro could be enhanced by subcloning the cell substrate prior to infection or by introducing multiple combinations of two or more mutations into the wild type genome. Various chimeric viruses induced liver enzyme elevations in marmosets, indicating that attenuation of virulence also required multiple mutations.

Animals↗

PubMed1983

Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA.

To test the capacity of poxviruses for added foreign DNA, a recombinant was constructed that contains 24 700 bp of bacteriophage lambda DNA inserted within the vaccinia virus thymidine kinase (TK) gene. The recombinant is stable, infectious and replicates in tissue culture at the same rate and to the same titer as standard vaccinia virus. This size flexibility of the poxvirus genome and the lack of stringent packaging requirements are useful features for an infectious eukaryotic cloning vector.

Bacteriophage lambda↗

PubMed1993

Consequences of gene transfer between distantly related tombusviruses.

Hybrid cDNA clones were constructed by fusing the coat protein-encoding gene and/or the 3'-terminal region (including the 22- and 19-kDa protein-encoding genes) derived from a clone of artichoke mottled crinkle tombusvirus to the 5'-terminal region of a full-length clone of cymbidium ringspot tombusvirus. In vitro transcripts from recombinant clones were infectious when inoculated into Nicotiana clevelandii plants. Inoculated plants showed symptoms different from those induced by parent viruses. In particular, systemic invasion depended very much, although not exclusively, on the type of protein that coated progeny viral RNA, suggesting a role of the capsid protein in the long-distance movement of tombusvirus infections.

Base Sequence↗

PubMed1993

HIV1 integrase expressed in Escherichia coli from a synthetic gene.

Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.

Amino Acid Sequence↗

PubMed1994

Generation of long flavivirus expression cassettes by in vivo recombination and transient dominant selection.

Assembly of expression cassettes coding for large segments of viral polyproteins is often complicated or impossible due to the instability of the resulting recombinant (re-) plasmids during propagation in Escherichia coli. Using the transient dominant selection approach described for the construction of vaccinia virus recombinants (re-VV), we have constructed several intermediate vectors and developed a procedure which enables direct assembly of long expression cassettes in the VV genome by in vivo recombination and does not require preliminary assembly of long cassettes in intermediate plasmids, thus eliminating the instability problems. The procedure was used to construct re-VV carrying fragments of the West Nile (WN), Murray Valley encephalitis (MVE), tick-borne encephalitis (TBE) and dengue type-2 (DEN2) viral genomes. Using this procedure, we have assembled a WN expression cassette which represents 86% of the WN genome and codes for 91% of its polyprotein and constitutes the longest flavivirus (FV) expression cassette inserted so far into the VV genome. Analysis of FV protein expression from the obtained recombinants indicates that recombination occurs with a high degree of specificity and the ORF remains intact. The procedure described offers a possible approach for the assembly of infectious cDNA clones.

Base Sequence↗

PubMed2006

Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein.

Safe and effective vaccination is important for rabies prevention in animals. Although several genetically engineered rabies vaccines have been developed, few have been licensed for use, principally due to biosafety concerns or due to poor efficacy in animal models. In this paper, we describe the construction and characterization of a replication-competent recombinant canine adenovirus type-2 expressing the rabies virus glycoprotein (SRV9 strain) by a different strategy from that reported previously, i.e., the recombinant genome carrying the glycoprotein cDNA was generated by a series of strictly gene cloning steps, infectious recombinant virus was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. This recombinant virus, CAV-E3delta-CGS, was subcutaneously injected into dogs. All vaccinated dogs produced effective neutralizing antibodies after one inoculation and a stronger anamnestic immune response was produced after booster injection. The immunized dogs could survive the challenge of 60,000 mouse LD50 CVS-24, which is lethal to all unimmunized dogs and is comparable to the conventional vaccines. The immunity lasts for months with a protective level of neutralizing antibody. This recombinant virus would be an alternative to the attenuated and the inactivated rabies vaccines and be prospective in immunizing dogs against rabies.

Adenoviruses, Canine↗

PubMed2004

Construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging pathogen causing significant economic losses in the swine industry worldwide. Two novel gene-deleted viruses were constructed and evaluated as vaccine candidates. Using the full-length infectious cDNA clone of North American PRRS isolate P129, the ORF2 and ORF4 genes (which encoded minor structural glycoproteins GP2a/2b and GP4, respectively) were individually deleted from the viral genome. Both deletion mutants were non-viable in MARC-145 cells and porcine alveolar macrophages, indicating that both genes are essential for virus replication. To rescue the replication-defective PRRSV, two complementing cell lines, MARC-2000 and MARC-400, were established to stably express the PRRSV GP2 and GP4 proteins, respectively. These cells were able to complement the deleted gene function of PRRSV in trans and supported production of the replication-defective DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses. Both DeltaORF2-PRRSV and DeltaORF4-PRRSV viruses were propagated for 40-50 generations in the corresponding complementing cells and remained replication-defective in MARC-145 cells. To examine the immunogenic potential of the replication-defective PRRSV as vaccine candidates, four groups of pigs, 20 pigs per group, were immunized twice with DeltaORF2-PRRSV or DeltaORF4-PRRSV and challenged with the homologous virulent virus at 3 weeks post-immunization. In spite of the fact one group showed significant reduction in virus load, we could not demonstrate improvement from clinical diseases in this vaccination/challenge study. However, we did show that the cDNA clone of PRRSV can be a useful tool to genetically engineer PRRSV vaccine candidates and to study pathogenesis and viral gene functions.

Animals↗

PubMed2004

An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus.

A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7_E2alf. After transfection of in vitro-transcribed CP7_E2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7_E2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7_E2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and "marker vaccine" properties of the generated chimeric CP7_E2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 10(7) TCID(50), CP7_E2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-E(RNS)-specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7_E2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7_E2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV "marker vaccine".

Animals↗

PubMed2004

Functional relationship between the matrix proteins of feline and simian immunodeficiency viruses.

To investigate the functional relationship between the matrix (MA) proteins of feline and simian immunodeficiency viruses (FIV and SIV, respectively), we generated chimeric proviruses in which the MA-coding region of an SIV infectious molecular clone was partially or fully replaced by its FIV counterpart. Chimeric SIV proviruses containing the amino-terminal 36 residues or the central and carboxy-terminal regions of the FIV MA assembled into virions as efficiently as wild-type SIV. However, the resulting virions were noninfectious in single-cycle infectivity assays. Furthermore, a chimeric SIV provirus containing the entire FIV MA was found to be severely impaired in virion production due to inefficient membrane binding of the chimeric Gag polyprotein. Interestingly, the assembly defective phenotype of this chimeric Gag precursor could be reversed either by introducing the G31K/G33K double amino acid substitution in the FIV-derived MA domain or by coexpression with wild-type SIV Gag. Of note, a chimeric FIV provirus expressing the SIV MA not only assembled into particles as efficiently as wild-type FIV, but also replicated in feline T cells with wild-type kinetics. Our results thus provide novel information about the functional homology between the MA proteins of distantly related lentiviruses.

Amino Acid Sequence↗

PubMed2005

The Bean pod mottle virus proteinase cofactor and putative helicase are symptom severity determinants.

Full-length infectious cDNA clones were constructed from the genomic RNAs of three distinct strains (K-G7, K-Ha1 and K-Ho1) of the comovirus Bean pod mottle virus (BPMV). Whereas K-G7, a subgroup I strain, and K-Ha1, a subgroup II strain produce mild mottling, the reassortant strain K-Ho1 (RNA1(I) + RNA2(II)) induces necrotic primary lesions on inoculated leaves of soybean and severe systemic leaf mottling and blistering. Pseudorecombinants of all possible combinations of transcripts were generated and tested for symptom production. Only soybean plants inoculated with combinations having RNA1 derived from the severe strain K-Ho1, regardless of the origin of RNA2, induced severe symptoms, indicating that symptom severity maps to RNA1. Experiments with chimeric RNA1 constructs indicated that the coding regions of the protease co-factor (Co-pro) and the C-terminal half of the putative helicase (Hel) are determinants of symptom severity. Symptom severity correlated well with higher accumulation of viral RNA, but neither the Co-pro nor Hel protein could be demonstrated as a suppressor of RNA silencing.

Base Sequence↗

PubMed2005

Conserved serines in simian immunodeficiency virus capsid are required for virus budding.

The simian immunodeficiency virus (SIV) capsid protein (CA), a constituent of the Pr55Gag polyprotein, is phosphorylated in virions but not in virus-producing cells (Rue, S.M., Roos, J.W., Tarwater, P.M., Clements, J.E., Barber, S.A., 2005. Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus. J. Virol. 79 (4), 2484-2492.). Using phosphoamino acid analysis of CA, we show that serine is the primary phosphate acceptor. A series of substitution mutants of serines in the CA domain of Pr55Gag were constructed in the infectious viral clone SIVmac239. These virus mutants were examined for defects in virus replication and virion infectivity, release, and morphology, as well as alterations in phosphorylation of CA-containing proteins. Although the virus mutants exhibited a number of replication defects, none of these defects could be directly attributed to aberrant CA phosphorylation. A novel defect was a block in early budding, which was common among several virus mutants with substitutions in the CA N terminus. Together, these results indicate that certain residues in the CA N terminus are crucial for early budding events.

Amino Acid Sequence↗