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PubMed1991

Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture.

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.

Animals↗

PubMed1989

Nucleotide sequence analysis of feline immunodeficiency virus: genome organization and relationship to other lentiviruses.

We determined the complete nucleotide sequence of an infectious proviral molecular clone (FIV-14) of the feline immunodeficiency virus (FIV). FIV-14 has a genome organization similar in complexity to other lentiviruses. In addition to three large open reading frames representing the gag, pol, and env genes, at least four small open reading frames are present in the pol-env intergenic, env, and env-3' long terminal repeat regions. Nucleotide and deduced amino acid sequence alignments of the FIV coding sequences with analogous sequences of other lentiviruses revealed significant identities only in the gag and pol genes. Phylogenetic tree analyses of gag and pol gene-encoded protein sequences demonstrate that FIV is more closely related to the ungulate lentiviruses, equine infectious anemia virus and visna virus, than to the primate lentiviruses, human and simian immunodeficiency viruses.

Animals↗

PubMed1995

Biological characterization and molecular cloning of murine C-type retroviruses derived from the TSZ complex from mainland China.

Characterization of the SRS murine retrovirus complex, derived from the TSZ system of murine leukemia developed in China, was carried out. The initial stock contained XC+, NB-tropic virus (and possibly other viruses), and induced several neoplastic diseases in neonatally inoculated NIH Swiss mice: erythroid leukemia, myeloid leukemia (acute myeloblastic leukemia), and lymphoblastic lymphoma (both B- and T-lymphoid). In addition, approximately 30% of inoculated animals developed central nervous system disease--hindlimb paralysis or semilateral paralysis. Rescue of virus from the spleen of an animal with combined erythroid/myeloid leukemia, followed by endpoint dilution gave two stocks: 19-6 (XC+, NB-tropic) and 19-7 (XC-, NB-tropic). Both stocks induced erythroid and myeloid leukemia, and 19-6 induced CNS symptoms as well. Southern blot analysis indicated that the predominant viruses from the 19-6 and the 19-7 cultures were related, but different in the env region. An infectious virus molecular clone of provirus from 19-6 cells was obtained. The resulting cloned virus [SRS 19-6 murine leukemia virus (MuLV)] induced four kinds of leukemia: erythroid, myeloid, B-lymphoma, and T-lymphoma; in many cases, more than one tumor type was identified in the same animal. Such a broad spectrum of leukemias induced by a cloned MuLV is unusual. Flaccid hindlimb paralysis induced by SRS 19-6 MuLV could be attributed to meningeal B-lymphoma. Immunofluorescent staining with a panel of env-specific monoclonal antibodies confirmed that the 19-6 and 19-7 viral stocks contained different viruses, which differed from previously characterized MuLVs. The viruses of the SRS complex may provide interesting reagents for investigations of MuLV-induced disease.

Animals↗

PubMed1995

In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids.

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA bacteriophage phi 29 per ml were assembled in vitro, with 11 proteins derived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a purified recombinant procapsid with the aid of a small viral RNA (pRNA) transcript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled capsids were subsequently converted into infectious virions after the addition of four more recombinant proteins for neck and tail assembly. Electron microscopy and genome restriction mapping confirmed the identity of the infectious phi 29 virions synthesized in this system. A nonstructural protein, gp13, was indispensable for the assembly of infectious virions. The overproduced tail protein gp9 was present in solution in mostly dimeric form and was purified to homogeneity. The purified gp9 was biologically active for in vitro phi 29 assembly. Higher-order concentration dependence of in vitro phi 29 assembly on gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to earlier findings for phages T4 and lambda. The work described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 assembly and dissection of functional domains of structural components, enzymes, and pRNA (C.-S. Lee and P. Guo, Virology 202:1039-1042, 1995). Efficient packaging of foreign DNA in vitro and synthesis of viral particles from recombinant proteins facilitate the development of phi 29 as an in vivo gene delivery system. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 29 carrying a tail fused to ligands for specific receptor of human cells.

Bacillus Phages↗

PubMed1983

Analysis of recombinant DNA clones of the endogenous BALB/c murine leukemia virus WN1802N: variation in long terminal repeat length.

We analyzed 15 recombinant DNA clones of the unintegrated closed circular DNA intermediate of the BALB/c endogenous ecotropic murine leukemia virus WN1802N. Thirteen of these clones had an insert which corresponded to the complete murine leukemia virus genome. Of these, six contained a single long terminal repeat (LTR) and seven contained two LTRs. The viral genomes in nine clones had an LTR of 520 base pairs (bp), one had an LTR of 570 bp, three had an LTR of 600 bp, and one had an LTR of 670 bp. Restriction endonuclease analysis demonstrated that the size variability resides in the U3 region. Seven of eight clones which yielded infectious virus by DNA transfection had the 520-bp LTR, and the other had a 600-bp LTR. More detailed examination of plasmid subclones of three isolates with different-sized LTRs revealed that the approximate position which varies in the U3 region corresponds to the 72-bp repeat region of Moloney sarcoma virus. Possible consequences of these variations are discussed.

Animals↗

PubMed1973

Persistent infection of cells in culture by measles virus. 3. Comparison of virus-specific RNA synthesized in primary persistent infection in HeLa cells.

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.

Carbon Isotopes↗

PubMed1997

A CaMV 35S promoter driven cDNA clone of tobacco mosaic virus can infect host plant tissue despite being uninfectious when manually inoculated onto leaves.

Leading from the success of inoculating plants with viral RNA transcribed in vitro from full length cDNA clones, attempts have been made to build cDNA clones which are directly infectious by inoculation. However, we and others have found that viral cDNA clones driven by the CaMV 35S promoter were able to infect some host plants yet not others, when manually inoculated onto leaves. Alternative methods including microprojectile bombardment have been used to deliver an infectious TMV construct into plant cells resulting in the infection of all TMV host plants tested. Lack of infection via manual inoculation may be due to unsuccessful delivery of a viable construct into the plant cell nucleus.

DNA, Complementary↗

PubMed1985

Infectivity studies on different potato spindle tuber viroid (PSTV) RNAs synthesized in vitro with the SP6 transcription system.

We have constructed two sets of clones in which one to six head-to-tail connected DNA copies of the potato spindle tuber viroid (PSTV) RNA genome were inserted into the plasmid pSP62- Pl downstream of the promoter for SP6 RNA polymerase. In vitro transcription of these constructs with the promoter-specific SP6 RNA polymerase yielded the corresponding oligomeric single-stranded linear PSTV RNA molecules of (+) and (-) polarity. Except for short vector-derived terminal sequences these in vitro synthesized PSTV RNA forms are equivalent to the RNA intermediates of the PSTV replication cycle which are present in vivo only in extremely low concentrations. From each DNA template molecule up to 600 RNA copies could be transcribed in vitro and yields > 100 microg were obtained. When mechanically inoculated to tomato seedlings the PSTV (+) RNA oligomers were as infectious as the natural PSTV (+) RNA monomers. Surprisingly, the corresponding oligomeric PSTV (-) RNAs wer approximately 10(4)-fold less infectious. However, when these (-) RNAs were partially protected prior to inoculation by mixing or hybridizing them with non-infectious (+) RNA fragments or by 'capping' their 5' terminus, an increase in the number of infections was observed. The in vitro synthesis of infectious RNA from cloned cDNA means that, in principle, it should be possible to develop vector systems from pathogens with RNA genomes.

Cloning, Molecular↗

PubMed1992

Peanut stunt virus satellite RNA: analysis of sequences that affect symptom attenuation in tobacco.

The V-satellite RNA (V-satRNA) of peanut stunt virus (PSV) has no effect on symptoms produced in tobacco by PSV. In contast, the G-satRNA induced complete or nearly complete suppression of systemic symptom development. Because G-satRNA differs from V-satRNA in only five nucleotide positions, these two satRNAs provide excellent material for investigating the molecular basis of satRNA-mediated symptom attenuation. For this purpose, we constructed transcription vectors containing full-length cDNA clones from which infectious RNA transcripts can be synthesized in vitro, and produced chimeric and mutant satRNA molecules. Although an A----C substitution at position 362 of the V-satRNA molecule delayed systemic symptom development and reduced symptom severity, changes at both nucleotide positions 226 (C----U) and 362 (A----C) of V-satRNA were required for suppression of systemic symptom development. Our results are consistent with the idea that PSV satRNAs are noncoding molecules that exert their biological activities by directly interacting with host/viral components.

Base Sequence↗

PubMed2002

The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome.

The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F' replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.

Animals↗

PubMed1999

Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution.

Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.

Animals↗

PubMed2007

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Animals↗

PubMed1998

The S2 gene of equine infectious anemia virus is dispensable for viral replication in vitro.

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAVUK. Various EIAVUK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAVUK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.

Amino Acid Sequence↗

PubMed1991

Transfer of the movement protein gene between two tobamoviruses: influence on local lesion development.

The effects of transfer of the movement gene between the tobamoviruses tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) were studied. The movement protein (MP) gene of TMGMV was cloned into an infectious cDNA of TMV to build the recombinant virus V23. V23, like TMV and TMGMV, caused systemic infection in Nicotiana tabacum Xanthi. In N. sylvestris V23 and TMV spread systemically although TMGMV produces necrotic local lesions on this host. V23 and TMV cause systemic infection on tomato plants while TMGMV does not infect tomato. In Xanthi nc plants, V23 produced necrotic local lesions similar in size to those produced by TMGMV. On the other hand in transgenic Xanthi nc tobacco plants that express a gene encoding the MP of TMV the necrotic lesions produced by V23 and TMGMV were similar in size to those produced by TMV. These results indicate that the size of necrotic lesions produced by TMGMV and TMV on Xanthi nc plants is influenced by the MP gene.

Amino Acid Sequence↗

PubMed1989

In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant.

A molecular clone of Venezuelan equine encephalitis virus (VEE) was constructed from four cDNAs that were synthesized using the viral RNA genome as template. Together, these cDNAs are believed to represent all but the nine 5'-terminal nucleotides of the VEE genome sequence. A T7 promoter, followed by a single intervening G residue, and the exact 5'-terminus of VEE were added to the 5'-most clone using in vitro mutagenesis. Appropriate restriction fragments isolated from the cloned cDNAs were joined to form a candidate full-length VEE cDNA clone. RNA transcripts synthesized in vitro from the cDNA clone were able to initiate a productive infection in DEAE-dextran-treated chicken embryo fibroblasts (CEF). VEE antigens were demonstrated in RNA-transfected cells, and supernatants from transfected cultures contained infectious virus particles. The candidate full-length cDNA clone lacked 102 nucleotides of the VEE genome sequence. The deletion, which also was present in the genomes of progeny virions derived from the clone, did not appear to affect growth in cultured CEF, baby hamster kidney cells, or Vero cells. The site of the deletion was mapped to the 3'-end of the nsP3 gene by comparison to other alphavirus sequences. In this region, the VEE genome sequence includes two tandem 102-nucleotide repeats which can be arranged in a stable stem and loop structure. The sequence remaining in the deleted clone retains one copy of the duplicated sequence and, in addition, faithfully preserves a portion of the predicted stem.

Antigens, Viral↗

PubMed2004

Reversion of molecularly engineered, partially attenuated, very virulent infectious bursal disease virus during infection of commercial chickens.

A molecularly cloned, tissue culture-adapted infectious bursal disease virus (BD-3tc) was generated from a very virulent strain by the reverse genetics approach following site-directed mutagenesis (Q253H and A284T in VP2). The pathogenicity of BD-3tc was tested in commercial chickens. The wild-type strain (BD-3wt) and the molecularly cloned parental strain (BD-3mc) were included for comparison. The subclinical course of the disease, with delayed and milder pathological lesions followed by quick follicular regeneration in the bursa of Fabricius in BD-3tc-inoculated birds, suggested that these amino acid substitutions made BD-3tc partially attenuated. However, severe bursa atrophy was observed at 14 days after inoculation. Reverse transcription-polymerase chain reaction coupled with restriction enzyme analysis revealed that both point mutations in BD-3tc had reverted 14 days after inoculation. Further investigations demonstrated that the codon for amino acid at position 284 had already reverted to the wild-type phenotype (T284A) 3 days after inoculation.

Animals↗

PubMed2002

Role of bovine viral diarrhea virus biotype in the establishment of fetal infections.

OBJECTIVE: To examine the role of bovine viral diarrhea virus (BVDV) biotype on the establishment of fetal infection in cattle. ANIMALS: 30 mixed-breed pregnant cows. PROCEDURE: Pregnant cows were inoculated oronasally with either i-WNADL, originating from an infectious BVDV cDNA clone of the National Animal Disease Laboratory (NADL) isolate, or the parental virus stock, termed NADL-A. RESULTS: All cows developed neutralizing antibodies to BVDV, and virus was commonly isolated from peripheral blood mononuclear cells or nasal swab specimens of NADL-A inoculated cows; however, virus was rarely isolated from specimens of i-WNADL inoculated cows. i-WNADL did not cause fetal infection, whereas all fetuses harvested from NADL-A inoculated cows at 6 weeks after inoculation had evidence of infection. Immunoblot analysis of fetal virus isolates revealed the absence of NS3, confirming a noncytopathic (NCP) biotype BVDV in the NADL-A stock. The sequence of the NCP contaminant (termed NADL-1102) and the i-WNADL genome were virtually identical, with the exception of a 270 nucleotide-long insert in the i-WNADL genome. Phylogenetic analyses revealed that NADL-1102 forms a monophyletic group with 6 other NADL genomes. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the contaminating NCP virus in the NADL-A stock was the ancestral NADL virus, which originally infected a bovine fetus and recombined to produce a cytopathic (CP) variant. Following oronasal infection of pregnant cows, viremia and transplacental transmission of CP BVDV to the fetus is rare, compared with the high occurrence of maternal viremia and fetal infection observed with NCP BVDV.

Animals↗

PubMed2002

Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene.

The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.

Amino Acid Sequence↗