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At least 433 records · Page 24Linked to original sources
PubMed2004

Isotropic-cholesteric phase transition of filamentous virus suspensions as a function of rod length and charge.

The viruses studied are genetically engineered, charged, semiflexible filamentous bacteriophages that are structurally identical to M13 virus, but differ either in contour length or surface charge. While varying contour length (L) we assume the persistence length (P) remains constant, and thus we alter the rod flexibility (L/P) . Surface charge is altered both by changing solution pH and by comparing two viruses, fd and M13, which differ only by the substitution of one charged for one neutral amino acid per virus coat protein. We measure both the isotropic and cholesteric coexistence concentrations as well as the nematic order parameter after unwinding the cholesteric phase in a magnetic field as a function of rod surface charge, rod length, solution ionic strength, and solution pH . The isotropic-cholesteric transition experimental results agree semiquantitatively with theoretical predictions for semiflexible, charged rods at high ionic strength, but disagree at low ionic strength.

Capsid↗
PubMed2004

Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif.

Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid. In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation. We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon. Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues. X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A. The foldon residues 458-483 also adopt their natural structure. The intervening linkers are not well ordered in the crystal structures. This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif. It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins. Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure. They can also prove useful for gene therapy and fibre engineering applications.

Adenoviridae↗
PubMed2000

Mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana.

Two different motifs in the coat protein (CP) of Plum pox virus (PPV) (R(3015)Q(3016), D(3059)) were mutated by replacing the respective amino acids with others possessing different chemical properties. The mutated CP genes were introduced into an infectious full-length clone of PPV (p35PPV-NAT) to investigate their influence on systemic infection of transgenic wild-type PPV CP-expressing and non-transgenic plants of Nicotiana benthamiana. All mutants failed to establish systemic infections in non-transgenic N. benthamiana plants, but were complemented by intact CP in transgenic plants. Moreover, the CP-RQ-D mutant (carrying mutations in both the RQ and D motifs) was introduced into p35PPV-NAT engineered to express beta-glucuronidase (GUS) for direct observation of systemic movement and particle assembly in N. benthamiana leaves. GUS-staining revealed that the CP mutant (RQ-D) was restricted to initially infected cells without forming virions. Systemic movement and particle assembly were restored in CP-transgenic N. benthamiana plants. Finally, transgenic N. benthamiana plants were generated that expressed each of the three mutated CP genes. Homozygous T(2) lines were selected and tested for resistance to PPV. Immunogold labelling and electron microscopy revealed that heterologous encapsidation with challenging Chilli veinal mottle virus and Potato virus Y was suppressed in these lines. In addition, assembly mutants did not complement CP-defective p35PPV-NAT. The possible use of modified viral CP genes for the production of virus-resistant transgenic plants, thereby reducing the putative risks of heterologous encapsidation and complementation, is discussed.

Capsid↗
PubMed1992

Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors.

The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.

Amino Acid Sequence↗
PubMed1992

Poliovirus chimeras expressing sequences from the principal neutralization domain of human immunodeficiency virus type 1.

Sequences from the principal neutralization domain of human immunodeficiency virus type 1 (HIV-1) strain LAI or RF have been expressed in antigenic site 1 of the capsid of the Sabin strain of poliovirus type 1. A number of the resulting chimeras were viable. Viable variants bearing mutations within the insertion site spontaneously arose from several nonviable chimeras. In general, these mutations result in a decrease in positive charge in the substituted antigenic site 1. Two of the chimeras were genetically stable and have been further characterized. Both chimeras were neutralized by various HIV-1 neutralizing antibodies. In rabbits, both chimeras produced high levels of antibodies which react with HIV-1 gp120/160 in immunoprecipitation and enzyme-linked immunosorbent assays. One of the chimeras (HIV-1LAI) produced a significant but weak HIV-1 neutralizing response.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Recombinant AAV-mediated gene delivery to the central nervous system.

Various regions of the brain have been successfully transduced by recombinant adeno-associated virus (rAAV) vectors with no detected toxicity. When using the cytomegalovirus immediate early (CMV) promoter, a gradual decline in the number of transduced cells has been described. In contrast, the use of cellular promoters such as the neuron-specific enolase promoter or hybrid promoters such as the chicken beta-actin/CMV promoter resulted in sustained transgene expression. The cellular tropism of rAAV-mediated gene transfer in the central nervous system (CNS) varies depending on the serotype used. Serotype 2 vectors preferentially transduce neurons whereas rAAV5 and rAAV1 transduce both neurons and glial cells. Recombinant AAV4-mediated gene transfer was inefficient in neurons and glial cells of the striatum (the only structure tested so far) but efficient in ependymal cells. No inflammatory response has been described following rAAV2 administration to the brain. In contrast, antibodies to AAV2 capsid and transgene product were elicited but no reduction of transgene expression was observed and readministration of vector without loss of efficiency was possible from 3 months after the first injection. Based on the success of pioneer work performed with marker genes, various strategies for therapeutic gene delivery were designed. These include enzyme replacement in lysosomal storage diseases, Canavan disease and Parkinson's disease; delivery of neuroprotective factors in Parkinson's disease, Huntington disease, Alzheimer's disease, amyotrophic lateral sclerosis, ischemia and spinal cord injury; as well as modulation of neurotransmission in epilepsy and Parkinson's disease. Several of these strategies have demonstrated promising results in relevant animal models. However, their implementation in the clinics will probably require a tight regulation and a specific targeting of therapeutic gene expression which still demands further developments of the vectors.

Animals↗
PubMed1993

[Mouse neurovirulence of antigenic chimeras (type I/II) of polioviruses].

The mouse-adapted Lansing strain of poliovirus type 2 PV-2(L) induces fatal poliomyelitis in mice after intracerebral inoculation, while mice inoculated with Mahoney strain of poliovirus type 1 PV-1(M) show no signs of disease. Previous work had indicated that both the 5' non-translated region of the viral genome and the viral capsid protein, neutralization antigenic site I (N-Ag1), were involved in mouse neurovirulence. In order to further explore the role of N-Ag1 in mouse neurovirulence, antigenic chimeras of two poliovirus strains, XF414 and XF324, were constructed. In the two strains, ten amino acids (in XF414) or 16 amino acids (in XF324) in antigenic site I in Vp1 of PV-I (M) were replaced with a PV-2(L)-specific sequences using a mutagenesis cartridge. Mouse neurovirulence tests indicated that mice cerebral inoculated with XF414 and XF324 developed poliomyelitis leading to paralysis or death. The viruses possessing antigenicity of the inoculating viruses were isolated from cerebral tissues of the paralyzed mice. The results demonstrated that N-Ag1 is an important determinant of poliovirus host range, and may be involved in attachment and penetration of poliovirus (in)to cells of the mouse central nervous system.

Animals↗
PubMed1995

Mutagenesis of the genome of mouse hepatitis virus by targeted RNA recombination.

Our laboratory has described a method for introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA. By using a thermolabile N protein mutant of MHV as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, engineered recombinant viruses were selected as heat-stable progeny resulting from cotransfection. We have recently reported an optimization of the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that recombinants can often be directly identified without employing a thermal selection. We present here a progress report on our use of this system to map MHV mutants and to construct N gene mutants which include (1) a mutant in which the internal open reading frame within the N gene (the I gene) has been disrupted, and (2) a series of recombinants in which portions of the MHV N gene have been replaced by the homologous regions from the N gene of bovine coronavirus. We also report on some mutants we have not been able to construct.

Amino Acid Sequence↗
PubMed2006

Packaging of brome mosaic virus subgenomic RNA is functionally coupled to replication-dependent transcription and translation of coat protein.

In Brome mosaic virus (BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an Agrobacterium-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4(Trans) and CP(Trans)) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4(Rep) and CP(Rep)), were mixed in all four pair-wise combinations and infiltrated to Nicotiana benthamiana leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus).

Bromovirus↗
PubMed1992

Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.

Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.

Amino Acid Sequence↗
PubMed1994

Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus.

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Alb1, maps to the N gene. Sequencing of the Alb1 N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Alb1 mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.

Amino Acid Sequence↗
PubMed2003

Cell-type specific targeting and gene expression using a variant of polyoma VP1 virus-like particles.

The variant VP1-Z of the polyomavirus coat protein VP1 has been recently described as an engineered fusion protein of VP1 and the antibody binding domain protein Z. This construct is able to specifically bind and functionally present antibodies on the surface of virus-like particles of VP1-Z. Here we demonstrate that with the binding of Herceptin, an antibody directed against the receptor tyrosine kinase ErbB2, a cell type-specific targeting was established. ErbB2-positive cell lines were transduced with different plasmids encoding eGFP or beta-galactosidase. With both reporter systems functional gene expression in transduced cells could be observed. The transduction was strictly dependent on the use of a ternary complex formed of VLPs of VP1-Z, Herceptin, and the reporter plasmid DNA. The use of single components or ErbB2-negative cell lines did not result in functional gene transfer. The transduction was also completely dependent on the use of chloroquine, a lysosomotropic reagent. This indicates that the complex is internalized by ErbB2-mediated endocytosis.

Antibodies, Monoclonal↗
PubMed1991

trans rescue of a mutant poliovirus RNA polymerase function.

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.

Capsid↗
PubMed1999

Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties.

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.

Animals↗
PubMed1997

Characterization of synthetic foot-and-mouth disease virus provirions separates acid-mediated disassembly from infectivity.

One of the final steps in the maturation of foot-and-mouth disease virus (FMDV) is cleavage of the VP0 protein to produce VP4 and VP2. The mechanism of this cleavage is unknown, but it is thought to function in stabilizing the virus particle and priming it for infecting cells. To investigate the cleavage process and to understand its role in virion maturation, we engineered synthetic FMDV RNAs with mutations at Ala-85 (A85) and Asp-86 (D86) of VP0, which border the cleavage site. BHK cells transfected with synthetic RNAs containing substitutions at position 85 (A85N or A85H) or at position 86 (D86N) yielded particles indistinguishable from wild-type (WT) virus in sedimentation and electrophoretic profiles. Viruses derived from these transfected cells were infectious and maintained their mutant sequences upon passage. However, BHK cells transfected with synthetic RNAs encoding Phe and Lys at these positions (A85F/D86K) or a Cys at position 86 (D86C) produced noninfectious provirions with uncleaved VP0 molecules. Despite their lack of infectivity, the A85F/D86K provirions displayed cell binding and acid sensitivity similar to those of WT virus. However, acid breakdown products of the A85F/D86K provirions differed in hydrophobicity from the comparable WT virion products, which lack VP4. Taken together, these studies are consistent with a role for soluble VP4 molecules in release of the viral genome from the endosomal compartment of susceptible cells.

Animals↗
PubMed2001

Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme.

Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of <10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.

Animals↗
PubMed1993

Efficient pathogen-derived resistance induced by integrated potato virus Y coat protein gene in tobacco.

The coat protein (CP) gene from potato virus Y (Hungarian isolate, PVY-H) was engineered into Agrobacterium tumefaciens binary vector for expression in different tobacco lines. Three different Nicotiana tabacum breeding lines were transformed and the integration of the CP gene was confirmed by PCR technique using genomic DNA preparations. The transcription and expression of the integrated CP gene was detected by Northern and Western blots. Pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of the transformed lines with purified PVY-H. The efficiency of protection varied between different transgenic plants ranging from almost complete to no protection. Five CP expressing tobacco lines were resistant to challenge infection with PVY-H as indicated by attenuation or absence of symptom development associated with reduction or lack of detectable virus accumulation. Data from Western blots showed that there is no correlation between the level of the expressed CP and the extent of protection. This suggests that the mechanism of the observed resistance is independent of the level of CP accumulation in the transgenic tobacco plants.

Agrobacterium tumefaciens↗
PubMed2006

Long-term persistence of gene expression from adeno-associated virus serotype 5 in the mouse airways.

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) have been used to deliver transgenes to the airways in a variety of pre-clinical and clinical studies. Gene transfer in these studies has been severely restricted by the basolateral localization of rAAV2 receptors. Here, we studied vectors constructed from the AAV5 genome and capsid, which utilize N-linked sialic acid-containing receptors found on the apical surface of airway epithelial cells. We investigated gene transfer efficacy and duration of transgene expression following delivery of rAAV5/5 vectors to the mouse respiratory tract. Robust, dose-dependent transgene expression was observed in the epithelium lining the nose for at least 32 weeks, and for at least 52 weeks in the lung. Importantly, in the lung, transgene expression mediated by rAAV5/5 was 40-fold greater than by rAAV2/2 vectors. A distinct cellular preference for rAAV5/5-mediated transduction was observed, with transgene expression being predominantly restricted to sustentacular cells of the olfactory epithelium in the nose and alveolar type II cells in the lung. Administration of rAAV5/5 vectors to both the nose and lungs led to the rapid development of rAAV5/5-neutralizing antibodies, suggesting that repeated administration may be severely hampered by host immune responses.

Animals↗