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PubMed1999

Establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons.

Defective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease N(pro) in addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7ins- (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding beta-glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7ins- does not induce cell lysis. Accordingly, neither N(pro) nor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7ins- with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV.

Animals↗

PubMed1999

Second-site reversion of a human immunodeficiency virus type 1 reverse transcriptase mutant that restores enzyme function and replication capacity.

Nonconservative substitutions for Tyr-115 in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) lead to enzymes displaying lower affinity for deoxynucleoside triphosphates (dNTPs) (A. M. Martín-Hernández, E. Domingo, and L. Menéndez-Arias, EMBO J. 15:4434-4442, 1996). Several mutations at this position (Y115W, Y115L, Y115A, and Y115D) were introduced in an infectious HIV-1 clone, and the replicative capacity of the mutant viruses was monitored. Y115W was the only mutant able to replicate in MT-4 cells, albeit very poorly. Nucleotide sequence analysis of the progeny virus recovered from supernatants of four independent transfection experiments showed that the Y115W mutation was maintained. However, in all cases an additional substitution in the primer grip of the RT (M230I) emerged when the virus increased its replication capacity. Using recombinant HIV-1 RT, we demonstrate that M230I mitigates the polymerase activity defect of the Y115W mutant, by increasing the dNTP binding affinity of the enzyme. The second-site suppressor effects observed were mediated by mutations in the 66-kDa subunit of the RT, as demonstrated with chimeric heterodimers. Examination of available crystal structures of HIV-1 RT suggests a possible mechanism for restoration of enzyme activity by the second-site revertant.

Animals↗

PubMed2000

Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity.

The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056-7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

Animals↗

PubMed2000

Productive replication of adeno-associated virus can occur in human papillomavirus type 16 (HPV-16) episome-containing keratinocytes and is augmented by the HPV-16 E2 protein.

We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.

Animals↗

PubMed2001

Requirement of cysteines and length of the human respiratory syncytial virus M2-1 protein for protein function and virus viability.

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.

Amino Acid Sequence↗

PubMed2002

Chimeric human immunodeficiency virus type 1 containing murine leukemia virus matrix assembles in murine cells.

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.

Animals↗

PubMed2002

Processing map and essential cleavage sites of the nonstructural polyprotein encoded by ORF1 of the feline calicivirus genome.

Feline calicivirus (FCV) nonstructural proteins are translated as part of a large polyprotein that undergoes autocatalytic processing by the virus-encoded 3C-like proteinase. In this study, we mapped three new cleavage sites (E(46)/A(47), E(331)/D(332), and E(685)/N(686)) recognized by the virus proteinase in the N-terminal part of the open reading frame 1 (ORF1) polyprotein to complete the processing map. Taken together with two sites we identified previously (E(960)/A(961) and E(1071)/S(1072)), the FCV ORF1 polyprotein contains five cleavage sites that define the borders of six proteins with calculated molecular masses of 5.6, 32, 38.9, 30.1, 12.7, and 75.7 kDa, which we designated p5.6, p32, p39 (NTPase), p30, p13 (VPg), and p76 (Pro-Pol), respectively. Mutagenesis of the E to A in each of these cleavage sites in an infectious FCV cDNA clone was lethal for the virus, indicating that these cleavages are essential in a productive virus infection. Mutagenesis of two cleavage sites (E(1345)/T(1346) and E(1419)/G(1420)) within the 75.7-kDa Pro-Pol protein previously mapped in bacterial expression studies was not lethal.

3C Viral Proteases↗

PubMed2002

Terminal RNA replication elements in human parechovirus 1.

To define structural elements critical for RNA replication in human parechovirus 1 (HPeV1), a replicon with chloramphenicol acetyltransferase as a reporter gene and an infectious virus cDNA clone have been used. It was observed that there are cis-acting signals required for HPeV1 replication located within the 5'-terminal 112 nucleotides of the genome and that these include two terminal stem-loops, SL-A and SL-B, together with a pseudoknot element. Significant disruption of any of these structures impaired both RNA replication and virus growth. In view of the similarity in terminal structures to several picornaviruses, such as cardioviruses and hepatoviruses, the insights generated in this work are of wider significance for understanding picornavirus replication.

5' Untranslated Regions↗

PubMed2003

Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: EAV virions contain six different envelope proteins. The glycoprotein GP(5) (previously named G(L)) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP(2b) (previously named G(S)), GP(3), and GP(4) are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP(5) and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP(2b), GP(3), and GP(4) proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP(2b) protein in the assembly of the GP(2b)/GP(3)/GP(4) complex. Open reading frames encoding cysteine-to-serine mutants of the GP(2b) protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP(2b) forms an intermolecular cystine bridge with one of the cysteines of the GP(4) protein, while the cysteine residues at positions 48 and 137 of GP(2b) are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP(4) protein is responsible for the covalent association of GP(3) with the disulfide-linked GP(2b)/GP(4) heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

Animals↗

PubMed2004

Human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles.

The human immunodeficiency virus type 1 (HIV-1) envelope comprises a surface gp120 and a transmembrane gp41. The cytoplasmic domain of gp41 contains cysteine residues (C764 and C837) which are targets for palmitoylation and were reported to be required for envelope association with lipid rafts and assembly on budding virions (I. Rousso, M. B. Mixon, B. K. Chen, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:13523-13525, 2000). Several infectious HIV-1 clones contain envelopes that have no gp41 cytoplasmic cysteines. Since no other gp41 amino acid is a target for palmitoylation, these clones imply that palmitoylation is not essential for envelope trafficking and assembly. Here, we show that HIV-1 envelope mutants that lack gp41 cytoplasmic cysteines are excluded from light lipid rafts. Envelopes that contained residues with bulky hydrophobic side chains instead of cysteines retained their association with heavy rafts and were nearly fully functional for incorporation into virions and infectivity. Substitution of cysteines with alanines or serines eliminated raft association and more severely reduced envelope incorporation onto virions and their infectivity. Nevertheless, the A764/A837 mutant envelope retained nearly 40% infectivity compared to the wild type, even though this envelope was excluded from lipid rafts. Our results demonstrate that gp41 cytoplasmic cysteines that are targets for palmitoylation and are required for envelope trafficking to classical lipid rafts are not essential for HIV-1 replication.

Cysteine↗

PubMed2004

Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2.

Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein.

Amino Acid Sequence↗

PubMed2004

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

Amino Acid Sequence↗

PubMed2006

Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides.

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 degrees C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid--histidine--serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.

Animals↗

PubMed2005

The role of reverse genetics systems in studying viral hemorrhagic fevers.

Viral hemorrhagic fever (VHF) is an infectious syndrome in humans often associated with high fatality rates. For most VHFs there are no specific and effective therapies or vaccines available and, in general, there is a lack of knowledge regarding the biology and pathogenesis of the causative agents. Therefore, a more detailed understanding of the molecular basis of VHF pathogenesis, including the identification of viral virulence determinants and host interactions and responses, will be important to enhance our ability to control VHF infections. The recently developed "reverse genetics systems" for several VHF causing viruses have allowed the generation of infectious viruses from cloned cDNA and thus, the generation of virus mutants. Here we review the existing reverse genetics systems for VHF causing viruses and discuss their use in studying viral replication, pathogenesis, and the development of antivirals and vaccines.

Animals↗

PubMed2004

Hepatitis C virus mutation affects proteasomal epitope processing.

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.

Amino Acid Sequence↗

PubMed2004

Spatial determinants of the alfalfa mosaic virus coat protein binding site.

The biological functions of RNA-protein complexes are, for the most part, poorly defined. Here, we describe experiments that are aimed at understanding the functional significance of alfalfa mosaic virus RNA-coat protein binding, an interaction that parallels the initiation of viral RNA replication. Peptides representing the RNA-binding domain of the viral coat protein are biologically active in initiating replication and bind to a 39-nt 3'-terminal RNA with a stoichiometry of two peptides: 1 RNA. To begin to understand how RNA-peptide interactions induce RNA conformational changes and initiate replication, the AMV RNA fragment was experimentally manipulated by increasing the interhelical spacing, by interrupting the apparent nucleotide symmetry, and by extending the binding site. In general, both asymmetric and symmetric insertions between two proposed hairpins diminished binding, whereas 5' and 3' extensions had minimal effects. Exchanging the positions of the binding site hairpins resulted in only a moderate decrease in peptide binding affinity without changing the hydroxyl radical footprint protection pattern. To assess biological relevance in viral RNA replication, the nucleotide changes were transferred into infectious genomic RNA clones. RNA mutations that disrupted coat protein binding also prevented viral RNA replication without diminishing coat protein mRNA (RNA 4) translation. These results, coupled with the highly conserved nature of the AUGC865-868 sequence, suggest that the distance separating the two proposed hairpins is a critical binding determinant. The data may indicate that the 5' and 3' hairpins interact with one of the bound peptides to nucleate the observed RNA conformational changes.

Alfalfa mosaic virus↗

PubMed2005

Pathogenesis and treatment of human T-cell leukemia virus infection.

The pathogenesis of human T-cell leukemia virus (HTLV)-induced adult T-cell leukemia-lymphoma (ATLL) was explored using an infectious molecular viral clone and a transgenic mouse model. Activation of nuclear factor-kappaB by the HTLV transcriptional transactivator protein Tax was found to be important for lymphocyte immortalization and tumorigenesis. Interferon-gamma regulates tumor development owing primarily to angiostatic effects. Translational clinical studies of chemotherapy, interferon-alpha, and nucleoside reverse transcriptase inhibitors have also assisted in identifying the pathogenic features of ATLL.

Animals↗

PubMed2001

Viral dynamics in hepatitis C.

The hepatitis C virus is an enveloped positive-sense single-stranded RNA virus, which has been classified into 6 major genotypes and over 100 subtypes. HCV replicates mainly in the hepatocyte. Recently, infectious HCV cDNA clones have been generated. Despite evidence that innate and adaptative humoral and cellular immune responses are activated as part of an antiviral defense, HCV has a remarkable ability to establish persistent infection. The analysis of viral kinetics using mathematical modeling shows a relative steady state without treatment, while an immediate biphasic HCV decline occurs in blood during successful treatment, the latter being predictive of clearance of HCV by the end of treatment.

Hepacivirus↗