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PubMed1989

Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication.

Sindbis virus (SIN) contains an in-frame opal termination codon in the nonstructural protein-coding region separating nsP3 and nsP4 and provides a useful tool to study the readthrough phenomenon of the termination codon in host cells and its role in viral replication. We have changed the opal codon by site-directed mutagenesis of a full-length SIN cDNA clone to either sense amino acids (serine, tryptophan, or arginine) or the other two translation termination codons (amber or ochre). Transcripts from all of the mutant cDNA clones were infectious when used to transfect chicken embryo fibroblasts. The resulting progeny virus stocks were then used to study the effects of these mutations on viral protein and RNA synthesis, growth properties, host range, and fitness compared with the parental strain. None of the mutants showed temperature sensitivity in plaquing efficiency or plaque morphology on chicken embryo fibroblast monolayers. Relative to the wild-type parent, the mutants containing sense replacements overproduced nsP34 but not nsP4 and made slightly decreased levels of nsP3, with a delay in its appearance. This indicates that the cleavage separating nsP3 and nsP4 occurs in these mutants and also that the level of nsP4 is not regulated solely by readthrough of the opal codon. The amber and ochre mutants produced decreased levels of nsP34, and the ochre mutant grew significantly more slowly than the other mutants or wild-type virus. For all five mutants, RNA synthesis early in infection was inhibited compared with that of the parental virus. This effect was apparent at multiplicities of infection of 20 PFU per cell but not at 100 PFU per cell. Using in situ hybridization to distinguish between mutant and wild-type plaques, we have studied the behavior of the serine mutant in a high-multiplicity growth competition experiment with wild-type virus. The wild-type virus eventually outcompeted the mutant after several passages, and these results indicate that this mutation has resulted in effects that are at least partially cis acting. Furthermore, by studying the growth, plaque formation, and protein synthesis of the mutants in various cell types, we have observed host range effects of the mutations, especially in mosquito and human cells. In addition, we have demonstrated, at least indirectly, that opal, amber, and ochre termination codons in the SIN nucleotide context can be suppressed in cultured cells of chicken, human, hamster, and mosquito origin.

Animals↗

PubMed2006

Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33.

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1-6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope.

Amino Acid Sequence↗

PubMed1988

Expression of a bacterial gene in plants mediated by infectious geminivirus DNA.

A viable coat protein deletion mutant of cassava latent virus (CLV) DNA 1 has been isolated, suggesting that this geminivirus might be exploited as a gene replacement vector. An extensive deletion of 727 nucleotides within the coat protein gene renders DNA 1 non-infectious. Chimeric clones have been constructed in which the deleted coat protein open reading frame has been replaced by the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene. Infectivity is restored to DNA 1 when the CAT gene is inserted in either orientation, producing symptoms typical of CLV infection. The results demonstrate that the coat protein plays no essential role in virus spread throughout the host. Levels of CAT expression of 80 U/mg soluble protein occur in systemically infected Nicotiana benthamiana leaves when the CAT gene is fused in-frame to the amino terminus of the coat protein, providing a sensitive assay for viral DNA replication.

Journal Article↗

PubMed1995

Molecular and serologic analysis in the transmission of the GB hepatitis agents.

Two flavivirus-like genomes have recently been cloned from infectious tamarin (Saguinus labiatus) serum, derived from the human viral hepatitis GB strain, which is known to induce hepatitis in tamarins. In order to study the natural history of GB infections, further transmission studies were carried out in tamarins. Reverse-transcription-polymerase chain reaction and enzyme-linked immunosorbant assays were developed for the detection of RNA and antibodies associated with the two agents, GB virus-A and GB virus-B. The infectivity of both of these agents was demonstrated in tamarins to be filterable through a 0.1 micron filter. Two distinct genomes were identified in the serum of eight of the infected tamarins, while in four tamarins, the genomes were detected independently of each other. Although specific antibodies to the GB virus-B epitopes were detected in the serum of animals inoculated with both agents or GB virus-B alone, antibodies to putative epitopes specific to GB virus-A were not detected in any of the animals. All tamarins inoculated with serum containing GB virus-B exhibited an elevation in liver enzyme levels after inoculation. Elevations of serum liver enzyme levels did not occur when GB virus-A was the only agent detected in the serum. Infection with the original infectious tamarin inoculum conferred protection from reinfection with GB virus-B but not with GB virus-A.

Animals↗

PubMed1993

Preliminary crystallographic analysis of bovine enterovirus.

Bovine enterovirus (BEV) strain VG-5-27 derived from an infectious cDNA clone has been crystallized as extended hexagonal plates. Virus recovered from crystals produced cytopathic effect in BHK cells. These crystals diffract X-rays from high energy synchrotron sources to beyond 2.7 A. The crystal system is monoclinic, space group P2(1) with unit cell dimensions a = 388 A, b = 390 A, c = 360 A, beta = 113 degrees. The virion is 300 A in diameter and one whole particle constitutes the crystallographic asymmetric unit, giving rise to 60-fold non-crystallographic redundancy.

Animals↗

PubMed1996

Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1.

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5' end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem-loop. In the guanine tetrad model interstrand guanine contacts from the dimer. We have made mutations preventing the dimerisation of subgenomic RNAs in vitro by these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1NL4-3 separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.

Antiporters↗

PubMed1999

Mutational analysis of Phe160 within the "palm" subdomain of human immunodeficiency virus type 1 reverse transcriptase.

The highly conserved Phe160 residue is located in the "palm" subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), and makes contact with Tyr115, a residue which is involved in deoxynucleoside triphosphate (dNTP) binding and fidelity of DNA synthesis. Five mutant RTs having Tyr, Trp, Ile, Ala or Gln instead of Phe160 were obtained by site-directed mutagenesis. F160Y and F160W retained substantial DNA polymerase activity, whereas the catalytic efficiency of nucleotide incorporation of mutants F160I, F160A and F160Q was less than 10 % that of the wild-type RT, using poly(rA).oligo(dT)20 as the template-primer. The low catalytic efficiency of mutants F160I, F160A and F160Q was due to their lower affinity for the dNTP substrate. F160Y displayed similar kinetic parameters as the wild-type RT in nucleotide insertion assays carried out with heteropolymeric DNA/DNA template-primers. However, nucleotide affinity was two- to sixfold reduced in the case of mutant F160W. Fidelity assays revealed similar misinsertion and mispair extension ratios for the three enzymes, although F160W showed a slightly higher accuracy of DNA synthesis, particularly in the presence of high concentrations of dNTP. When introduced in an infectious proviral clone, mutations F160I, F160A and F160Q rendered non-viable virus. The importance of Phe160 for polymerase function and viral replication could be mediated by its interaction with Tyr115, as suggested by the analysis of the available crystal structures of HIV-1 RT.

Amino Acid Substitution↗

PubMed1993

2B and 2C mutations are essential but mutations throughout the genome of HAV contribute to adaptation to cell culture.

Chimeric viruses constructed from various portions of two infectious cDNA clones representing the genomes of the wild-type and cell culture-adapted mutants of the HM-175 strain of hepatitis A virus were compared for their ability to replicate in cultures of fetal rhesus kidney cells. Mutations located in either the 5' or 3' third of the genome could markedly enhance growth in vitro but only when they were combined with mutations in the P2 region within either the 2B or the 2C gene. Therefore, mutations in 2B and 2C are essential for cell culture adaptation but mutations elsewhere in the genome also contribute significantly to the enhanced growth rate.

Adaptation, Biological↗

PubMed1994

Involvement of the cucumber necrosis virus coat protein in the specificity of fungus transmission by Olpidium bornovanus.

Cucumber necrosis (CNV) and the cherry strain of tomato bushy stunt (TBSV-Ch) are tombusviruses which differ in transmissibility by the fungus Olpidium bornovanus (Sahtiyanci) Karling (= O. radicale Schwartz and Cook). Zoospores acquire and transmit CNV, but not TBSV-Ch, in the in vitro manner. To assess the role of the coat protein in the specificity of fungus transmission, reciprocal exchanges were made between the coat protein genes of these two viruses in full-length infectious cDNA clones. Virions containing a modified TBSV-Ch genome with the CNV coat protein gene were efficiently transmitted, but those containing a modified CNV genome with the TBSV-Ch coat protein gene were not. This is the first direct demonstration for the role of a viral coat protein in the specificity of transmission by a fungus.

Capsid↗

PubMed1995

Differences in feline immunodeficiency virus host cell range correlate with envelope fusogenic properties.

Feline immunodeficiency virus (FIV) establishes persistent infections in cats inducing an acquired immunodeficiency syndrome. Differences in cell tropism have been observed among isolates of FIV (T. R. Phillips et al., J. Virol. 64, 4605-4613, 1990). The progeny of the infectious molecular clone of FIV p34TF10 was able to productively infect a feline fibroblast cell line, Crandell feline kidney cell, (CrFK), while the progeny of the molecular clone pPPR was not. However, pPPR, after transfection of CrFK cells, did produce virions which were able to productively infect feline lymphocytes. To analyze the mechanisms responsible for such differences in tropism and particularly the role of the envelope glycoproteins (Env), Env expression vectors were constructed by deletion of gag and pol genes from 34TF10 and PPR proviral clones. Env expression and function were studied by using a syncytium-formation assay and a quantitative ELISA. After transfection of CrFK, both 34TF10 and PPR Env precursors were correctly processed and Env surface glycoprotein, gp100, was released in culture supernatants. However, the Env of 34TF10 caused a dramatic syncytial effect in CrFK cells, while PPR Env did not induce any syncytium formation. The Env of 34TF10 placed under the control of the long terminal repeat of PPR maintained its ability to induce CrFK fusion. These results suggest that the inability of FIV PPR to infect CrFK fibroblasts is related to a restriction of virus entry mediated by the viral envelope.

Animals↗

PubMed1995

Molecular analysis of the essential and nonessential genetic elements in the genome of peanut chlorotic streak caulimovirus.

The DNA genome of caulimoviruses contains a set of essential genes: I (movement gene), IV (major capsid protein gene), V (reverse transcriptase gene), and VI (gene coding for a post-transcriptional activator of the expression of other virus genes). In peanut chlorotic streak caulimovirus (PCISV), three ORFs, A, B, and C, are located between genes I and IV. They are dissimilar to other caulimovirus ORFs. ORF VII of PCISV is a homolog of ORF VII of soybean chlorotic mottle caulimovirus (SoCMV), but is not similar to the nonconserved ORF VII in other caulimoviruses. The sequence complementary to a portion of tRNA(Met), thought to be essential for the priming of minus-strand DNA synthesis in caulimoviruses, is located within the coding sequence of ORF A. To explore the functional significance of ORFs VII, A, B, and C, various mutations were engineered into an infectious DNA clone of PCISV. ORFs VII and B are shown to be dispensable, while ORFs A and C are essential. ORF C is a possible functional equivalent of gene III in other caulimoviruses. Sequences within ORF A that are required for efficient priming of minus-strand synthesis are likely to extend beyond the 12-bp tRNA-binding site. Complete deletion of ORF VII was correlated with severe symptoms, notably with the necrosis of apical meristems. Significance of these observations for the understanding of replication and pathogenesis of plant pararetroviruses and for the improvement of caulimovirus-based expression vectors is discussed.

Arachis↗

PubMed1995

The tomato bushy stunt virus replicase proteins are coordinately expressed and membrane associated.

Two open reading frames at the 5'-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92-kDa (p92) readthrough product. From amino acid sequence comparisons with other small single-stranded RNA viruses, these proteins resemble viral components of the replicase-transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the carboxy-terminal "half" of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 readthrough protein, but the anti-p92 antibody was specific for p92. Immunoblot analyses revealed that at an early stage of infection both proteins were associated with the membrane fractions isolated from virus-infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic fraction. At all time points in plants and protoplasts p33 was about 20-fold more abundant than p92. A series of mutations derived from an infectious cDNA clone demonstrated that both the p33 and the p92 proteins were required for replication in protoplasts and the ratio of the two proteins was maintained in the replication-competent mutants. The wild-type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants (UAC or UAU) that were predicted to express only p92 were not viable in protoplasts.

Animals↗

PubMed1995

Characterization of sequences controlling the synthesis of alfalfa mosaic virus subgenomic RNA in vivo.

RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the viral coat protein (CP). CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants. In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity. This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.

Alfalfa mosaic virus↗

PubMed1995

Identification of hypervariable and conserved regions in the surface envelope gene in the bovine lentivirus.

The surface envelope (SU) gene of nine different isolates of the bovine lentivirus (BIV) were compared for nucleotide and deduced amino acid (aa) sequence diversity. Analyses were done both on isolates derived from the original reference strain, R29, and on field isolates of BIV. Six conserved and six hypervariable regions were identified. Many of the hypervariable regions were located in areas predicted to be on the surface of the SU protein. The SU gene comparison among all isolates showed up to a 50% aa sequence divergence. When a conserved region of the reverse transcriptase gene was compared among eight of the isolates, there was less than 11% aa sequence divergence. When comparing all isolates, the greatest size differences in the SU gene are observed in the 2nd hypervariable region (V2) with up to a 104-aa difference between the largest and smallest variant. R29-106, an infectious molecular clone of the original isolate of BIV, has an 87-bp deletion in V2 as compared to prototype isolate R29-127. All R29-derived isolates sequenced for this study had a SU gene size similar to R29-106. The four field isolates sequenced for this study had SU genes larger than R29-127. R29-derived isolates may not be representative of BIV currently present in United States cattle.

Amino Acid Sequence↗

PubMed1996

Influence of ORF2 on host cell tropism of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a lentivirus associated with an immunodeficiency syndrome of the domestic cat. A short open reading frame (ORF2), of unknown function, is present in all FIV isolates. We have investigated the role of ORF2 in determining the cell tropism of two infectious molecular clones of FIV. FIV-PPR is able to productively infect feline peripheral blood leukocytes (PBLs) and a T lymphocyte cell line (MCH5-4), but not a feline astrocyte cell line (G355-5) or Crandell feline kidney cells (CrFK). In contrast, FIV-34TF10 is able to productively infect G355-5 and CrFK cells, but not PBLs or MCH5-4 cells. The major difference in these FIV clones is that ORF2 in FIV-PPR is capable of encoding a 79-amino-acid peptide, whereas there is a stop codon in ORF2 after 43 amino acids in FIV-34TF10. We performed site-directed mutagenesis to change the stop codon (TGA) in FIV-34TF10 to a tryptophan (TGG), the amino acid present at this location in FIV-PPR. FIV-34TF10 with ORF2 repaired (FIV-ORF2rep) productively infected PBLs, MCH5-4 cells, and primary macrophages, as well as CrFK and G355-5 cells, indicating that a protein encoded by ORF2 plays a role in determining the host cell tropism of FIV. ORF2 contains hydrophobic, acidic, and leucine-rich domains similar to those shown to be important for transactivating proteins of other lentiviruses. Coexpression of a plasmid expressing the ORF2 gene product with another construct expressing the chloramphenicol acetyl transferase (CAT) gene driven by the FIV LTR, resulted in transactivation of CAT expression in both feline and human cells.

Amino Acid Sequence↗

PubMed1996

Conservation of a hairpin ribozyme sequence in HIV-1 is required for efficient viral replication.

We have previously described a hairpin ribozyme that targets a highly conserved sequence in the U5 region of HIV-1. To determine if escape mutations would compromise virus replication, we introduced critical mutations into the ribozyme target site of an infectious molecular clone of HIV-1MN. HIV-1 MNA has a substitution of A for G immediately 3' to the cleavage site and HIV-1 MNGC has two substitutions in the flanking sequences that are complementary to the ribozyme. In vitro studies confirmed that neither the MNA-nor the MNGC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MNGC-mutated target sequence failed to bind the ribozyme. Compensatory GC substitutions in the substrate recognition domain of the ribozyme resulted in a switch of binding and cleavage specificity. Replication of both the MNA and MNGC mutant viruses was initially two to three logs lower than that of wild-type virus, but after 3 weeks, virus production rose sharply in both cultures. Nucleotide sequence of RT-PCR-amplified viral sequences obtained from virus produced at later time points revealed complete reversion of MNA or partial reversion of MNGC to wild-type genotypes. No additional mutations within the ribozyme target sequence were observed. These results indicate that mutations in this conserved ribozyme target sequence led to significant attenuation of HIV-1MN.

Animals↗

PubMed1997

Gene transfer using replication-defective human foamy virus vectors.

Replication-defective vectors based on an infectious molecular clone of human foamy virus (HFV) were constructed by deletion and replacement of the accessory genes with expression cassettes for puromycin-resistance and beta-glucouronidase. Cell lines which produced in excess of 10(5) helper virus-free transducing units/ml were generated by trans-complementation of the replication defect using a BHK-21-derived cell line expressing the Bel-1 transactivator. Vectors based on the HFV genome may provide useful alternatives to existing retroviral vectors.

Animals↗

PubMed1997

Maintenance of an unusual polypurine tract in HIV-2: stability to passage in culture.

A stretch of purine residues, the polypurine tract (PPT), is found in all retroviruses and is used to initiate plus-strand DNA synthesis. While the PPT of most lentiviruses is a homogeneous sequence of purine residues, the PPT of some isolates of the human and simian immunodeficiency viruses is interrupted with a single pyrimidine residue. The ROD strain of human immunodeficiency virus type 2 (HIV-2) has such a pyrimidine-containing variant PPT. Virus generated from an infectious molecular clone, pROD10, was used to infect two CD4-positive T-cell lines, H9 and CEM. The sequence of the PPT was determined after two passages. From both cell lines, the variant PPT was retained, demonstrating that the presence of a pyrimidine in the PPT was fully functional and that there was no strong selection for an all-purine PPT.

Amino Acid Sequence↗