Replication Competence Research Articles

Mutations within the 5' nontranslated region of hepatitis A virus RNA which enhance replication in BS-C-1 cells.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/p16). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous African green monkey kidney (BS-C-1) cells, indicating that 5'NTR mutations in HM175/p35 virus are required for optimal growth in these cells. A chimera with the 5'NTR sequence of HM175/p16 retained the large foci of HM175/p35 virus, while the growth properties of other viruses having chimeric 5'NTR sequences indicated that mutations at bases 152 and/or 203 to 207 enhance replication in BS-C-1 cells. These findings were confirmed in one-step growth experiments, which also indicated that radioimmunofocus size is a valid measure of virus replication competence in cell culture. An additional mutation at base 687 of HM175/p16 had only a minor role in enhancing growth. In contrast to their effect in BS-C-1 cells, these 5'NTR mutations did not enhance replication in continuous fetal rhesus monkey kidney (FRhK-4) cells. Thus, mutations at bases 152 and/or 203 to 207 enhance the replication of HAV in a highly host cell-specific fashion.

Characterization of infectious and defective cloned avian hepadnavirus genomes

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.

In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region

Hepatitis B virus (HBV) variants with a nonsense mutation in the distal pre-C region have been detected in patients positive for anti-H Be, and the complete nucleotide sequence of one cloned pre-C variant has been determined. Transfection of this HBV variant clone into the human hepatoma cell line HepG2 resulted in the appearance of major HBV transcripts, replicative forms of viral DNA evidenced by both molecular hybridization and endogenous DNA polymerase assay, as well as the expression and secretion of HBcAg and HBcAg particles. Western blotting revealed only the 21-kDa HBcAg but not the 17-kDa HBeAg. These results demonstrate the replication capacity of the HBV variant with a nonfunctional pre-C region despite its inability to express HBeAg.

Mutagenesis of the 3' nontranslated region of Sindbis virus RNA

A cDNA clone from which infectious RNA can be transcribed was used to construct 42 site-specific mutations in the 3' nontranslated region of the Sindbis virus genome. The majority of these mutations were made in the 3'-terminal 19-nucleotide conserved sequence element and consisted of single nucleotide substitutions or of small (1 to 8) nucleotide deletions. An attempt was made to recover mutant viruses after transfection of SP6-transcribed RNA into chicken cells. In most cases, viable virus was recovered, but almost all mutants grew more poorly than wild-type virus when tested under a number of culture conditions. In the case of mutations having only a moderate effect, the virus grew as well as the wild type but was slightly delayed in growth. Mutations having a more severe effect led to lower virus yields. In many cases, virus growth was more severely impaired in mosquito cells than in chicken cells, but the opposite phenotype was also seen, in which the mutant grew as well as or better than the wild type in mosquito cells but more poorly in chicken cells. One substitution mutant, 3NT7C, was temperature sensitive for growth in chicken cells and severely crippled for growth in mosquito cells. Insertion mutations were also constructed which displaced the 19-nucleotide element by a few nucleotides relative to the poly(A) tail. These mutations had little effect on virus growth. Deletion of large regions (31 to 293 nucleotides long) of the 3' nontranslated region outside of the 19-nucleotide element resulted in viruses which were more severely crippled in mosquito cells than in chicken cells. From these results, the following principles emerge. (i) The entire 3' nontranslated region is important for efficient virus replication, although there is considerable plasticity in this region in that most nucleotide substitutions or deletions made resulted in viable virus and, in some cases, in virus that grew quite efficiently. Replication competence was particularly sensitive to changes involving the C at position 1, the A at position 7, and a stretch of 9 U residues punctuated by a G at position 14. (ii) The panel of mutants examined collectively deleted the entire 3' nontranslated region. Only mutants in which 8 nucleotides in the 3' terminal 19 nucleotides had been deleted or in which the 3' terminal C was deleted were nonviable. Although the 3' terminal C was essential for replication, it could be displaced by at least 7 nucleotides from its 3' terminal position adjacent to the poly(A) tract.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversion of Autonomously Replicating Sequence Mutations in Saccharomyces cerevisiae: Creation of a Eucaryotic Replication Origin within Procaryotic Vector DNA

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.

Molecular and biological characterization of a replication competent human immunodeficiency type 2 (HIV-2) proviral clone.

We obtained complete genomic clones of human immunodeficiency virus type 2 (HIV-2) from the DNA of the neoplastic human cell line HUT 78 freshly infected with a HIV-2 isolate, strain SBL6669. The recombinant phage DNA was transfected into the lymphocytes of CD4-positive HUT 78 cell line to test the replication competence of the proviral DNA. One genomic clone, designated HIV-2SBL/ISY, yielded retroviral particles after a few weeks of culture of the transfected cells. The HIV-2SBL/ISY clone contained a complete provirus and cellular flanking sequence. We obtained the DNA sequence of the provirus and compared it with the published sequence of two other HIV-2 isolates. The degree of variability among HIV-2 isolates is comparable to that observed among African HIV-1 isolates sequenced to date. Immunologically, HIV-2SBL/ISY is similar to the parental virus (HIV-2SBL6669) but differs in the envelope transmembrane protein that is truncated (gp32-34) in the parental virus and not in HIV-2SBL/ISY (gp41). Both the parental and the cloned viruses are infectious and cytopathic for some human T-cell lines, induce syncytia, and infect a human macrophage cell line (U937) in vitro. The availability of a biologically active HIV-2 clone provides the means to study the role and interaction of HIV-2 genes in vitro as well as to assess the functional similarities among HIV-1 and HIV-2 genes. Since HIV-2SBL/ISY cloned virus infects fresh peripheral blood T cells from Rhesus macaques in vitro and infects the same animal in vivo, its use in animals may represent a model for functional study of viral genes in vivo as well as for development of experimental approaches to prevent and cure retroviral infection in humans.

Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen

The efficiency of replication of plasmids containing the control region of polyomavirus DNA including one, two, or all three of the strong binding sites for large T antigen was measured in COP 8 cells which provide polyomavirus T antigen in trans. It was found that plasmids carrying only binding site A (the one closest to the origin core region) exhibited only 10% of the replication competence of plasmids with binding sites A and B or A and C. Plasmids containing all three binding sites, A, B, and C, did not replicate more efficiently than those with only two strong T-antigen-binding sites. We conclude, therefore, that optimal T-antigen-dependent replication of polyomavirus DNA requires two high-affinity T-antigen-binding sites.

Molecular mechanisms of regulation of HTLV-1 gene expression and its association with leukemogenesis.

Human T-cell leukemia virus type 1 (HTLV-1) has two trans-acting regulator genes, tax and rex, in the pX region. The tax gene is a trans-acting transcriptional activator of the long terminal repeat (LTR) and also of the cellular gene for IL-2R alpha. The latter seems to explain initiation of abnormal growth of HTLV-1 infected cells. The rex gene is a posttranscriptional regulator accumulating gag and env mRNA and also indirectly suppressing the transcription. The regulation requires two cis-acting elements, the LTR sequence at the 3' terminus and 5' splice signal, suggesting a novel mechanism of RNA processing in the nucleus. These two trans-activator genes are essential for efficient replication of HTLV-1 and also explain its poor replication competence and tendency to be latent in vivo.

Mammalian DNA polymerase alpha: a replication-competent holoenzyme form from calf thymus.

Calf thymus DNA polymerase alpha, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli, can be isolated as a distinct complex. A specific multiprotein form of the polymerase alpha, a form designated replication-competent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme alpha selectively initiates PCV DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.

NON-CYTOCIDAL NATURAL VARIANTS OF HUMAN IMMUNODEFICIENCY VIRUS ISOLATED FROM AIDS PATIENTS WITH NEUROLOGICAL DISORDERS

To understand the mechanism of HIVmediated neuropathology five viral isolates were obtained from four AIDS cases with central nervous system manifestations as the primary involvement. The isolates were identified as HIVs by antigenic cross-reactivity and nucleic acid hybridisations to HIV-specific antibodies and DNA probes. The replication and cytopathic properties of these isolates were studied and compared with lymphadenopathy-associated virus (HIVLAV). All isolates had replication competence equivalent to LAV, but four isolates did not kill T4 (CD-4) cells. This isolation of non-cytocidal natural variants of HIV raises the possibility that in some AIDS cases the neurological disorders might be due to HIV variants that are non-cytocidal to T4 cells. The results also indicate that virus replication and cytotoxicity are not always concordant functions in HIV.

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.

Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.