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PubMed1994

Replication of a Bacillus subtilis oriC plasmid in vitro.

We constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis-elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi-conservative and only one round of replication occurred within 15 min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2-dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and undirectional replication occurred in vitro.

Bacillus subtilis↗
PubMed1995

Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy.

The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC to the pT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.

Bacterial Proteins↗
PubMed1973

Bi-directional chromosomal replication in Salmonella typhimurium.

Transducing frequencies of phage P22 lysates prepared from Salmonella typhimurium exponential cultures in minimal and nutrient broth media were compared. The assumption is that cells grown in a minimal medium will have one replication fork per replication unit, but cells in nutrient broth will have multiple replication forks; therefore, the frequency of genetic markers near the origin of replication will be higher in the nutrient broth culture. Analysis of transduction showed a gradient of marker frequencies from the highest (the cysG-ilv region) to the lowest (purE-trpB region) in both clockwise and counter clockwise directions. This supports our previous observation that chromosome replication proceeds bidirectionally from the origin between cysG (109 min on S. typhimurium map) and ilv (122 min) to a terminus in purE-trpB region (20 to 53 min). Since this method avoids possible artifacts of other methods, the results are assumed to reflect the sequence of chromosome replication in exponentially growing cells. Evidence for the existence of multiple replication forks in nutrient broth-grown cells was supported by the following: (i) the marker frequency data fitted the assumption of multiple replication fork formation; (ii) residual deoxyribonucleic acid increase after inhibition of protein synthesis to complete a round of chromosome synthesis which was 44% in cells grown in a minimal medium and 82% in those in nutrient broth; (iii) segregation patterns of the (3)H-thymidine-labeled chromosome strands during subsequent growth in non-radioactive medium were studied by autoradiography, and the number of replication points per chromosome per cell was estimated as 5.6 for the nutrient broth culture and 2.5 for the minimal medium culture. These data support a model of symmetrical and bidirectional chromosome replication.

Autoradiography↗
PubMed1982

Origin and direction of mini-R1 plasmid DNA replication in cell extracts of Escherichia coli.

Replication of the mini-R1 plasmids pKN177 and pKN182 can be carried out efficiently in cell extracts of Escherichia coli and depends on both transcription and translation. Heteroduplex and restriction analyses indicate that both plasmids are derived from the R1 copy mutant pKN104 by Is1-mediated recombination events without involving structural alterations in the replication region. To ascertain whether the in vitro replication of these miniplasmids corresponds to R1 replication in vivo, the origin and direction of replication were analyzed by electron microscopy of replicative intermediates. It was found that replication starts at a unique origin located within the RepA region at R1 coordinate at 82.4 kilobases and proceeds unidirectionally toward the IS/b sequence. The specification of the origin and the direction of in vitro replication are therefore in full agreement with the pattern observed previously for the in vivo replication of the closely related plasmids R100 and R6-5. This agreement provides additional evidence that R1 DNA synthesis in vitro employs the same replication mechanism as it does in vivo.

Base Sequence↗
PubMed1999

Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function.

After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene. In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication.

Adenosine Triphosphatases↗
PubMed2006

Relationships between hepatitis C virus replication and CXCL-8 production in vitro.

The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.

Cell Line, Tumor↗
PubMed1973

Multiple and specific initiation of T4 DNA replication.

Partially replicated T4 DNA molecules (PRM) whose parental or progeny DNA was labeled with bromodeoxyuridine BUdR was analyzed by gradual shearing followed by CsCl banding of the sheared product. Analysis of PRM containing 18-mum replicated DNA showed that each replicated region was 3- to 6-mum long, indicating three to 6 replicative sites per molecule. Analysis of PRM containing 9-mum replicated DNA similarly indicated two to three replicated regions per molecule. DNA from the replicated regions of PRM containing 10-mum replicated DNA ("donor") was hybridized to DNA from mature phage ("recipient"), and the resulting hybrid was subjected to digestion with exonuclease I. The extent of protection of the recipient and more efficient self-annealing of progeny fragments from PRM indicated that the replicated regions represented 8 to 10 nonrandom locations of the genome. Possible significance of multiple sites for initiation of DNA replication is discussed.

Bromodeoxyuridine↗
PubMed1984

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.

Animals↗
PubMed1991

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

Animals↗
PubMed1992

trans-acting requirements for replication of Epstein-Barr virus ori-Lyt.

Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.

Amino Acid Sequence↗
PubMed1994

Immunization with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.

Animals↗
PubMed1994

Papillomavirus contains cis-acting sequences that can suppress but not regulate origins of DNA replication.

Bovine papillomavirus (BPV) DNA has been reported to restrict its own replication and that of the lytic simian virus 40 (SV40) origin to one initiation event per molecule per S phase, which suggests BPV DNA replication as a model for cellular chromosome replication. Suppression of the SV40 origin required two cis-acting BPV sequences (NCOR-1 and -2) and one trans-acting BPV protein. The results presented in this paper confirm the presence of two NCOR sequences in the BPV genome that can suppress polyomavirus (PyV) as well as SV40 origin-dependent DNA replication as much as 40-fold. However, in contrast to results of previous studies on SV40, most of the suppression of the PyV origin was due to NCOR-1, a 512-bp sequence that functioned independently of distance or orientation with respect to the PyV origin and that was not required for BPV DNA replication. Moreover, NCOR-1 alone or together with NCOR-2 did not restrict the ability of the PyV ori to reinitiate replication within a single S phase and did not require any BPV protein to exert suppression. Furthermore, NCOR-1 did not suppress BPV origin-dependent DNA replication except in the presence of PyV large tumor antigen (T-ag). Since NCOR-1 suppression of PyV origin activity also varied with T-ag concentration, suppression of origins by NCOR sequences appeared to require papovavirus T-ag. Therefore, it is unlikely that NCOR sequences are involved in regulating BPV DNA replication. When these results are taken together with those from other laboratories, BPV appears to be a slowly replicating version of papovaviruses rather than a model for origins of DNA replication in eukaryotic cell chromosomes.

Antigens, Viral, Tumor↗
PubMed1995

Sequences flanking the pentanucleotide T-antigen binding sites in the polyomavirus core origin help determine selectivity of DNA replication.

Replication of the genomes of the polyomaviruses requires two virus-specified elements, the cis-acting origin of DNA replication, with its auxiliary DNA elements, and the trans-acting viral large tumor antigen (T antigen). Appropriate interactions between them initiate the assembly of a replication complex which, together with cellular proteins, is responsible for primer synthesis and DNA chain elongation. The organization of cis-acting elements within the origins of the polyomaviruses which replicate in mammalian cells is conserved; however, these origins are sufficiently distinct that the T antigen of one virus may function inefficiently or not at all to initiate replication at the origin of another virus. We have studied the basis for such replication selectivity between the murine polyomavirus T antigen and the primate lymphotropic polyomavirus origin. The murine polyomavirus T antigen is capable of carrying out the early steps of the assembly of an initiation complex at the lymphotropic papovavirus origin, including binding to and deformation of origin sequences in vitro. However, the T antigen inefficiently unwinds the origin, and unwinding is influenced by sequences flanking the T antigen pentanucleotide binding sites on the late side of the viral core origin. These same sequences contribute to the replication selectivity observed in vivo and in vitro, suggesting that the inefficient unwinding is the cause of the replication defect. These observations suggest a mechanism by which origins of DNA replication can evolve replication selectivity and by which the function of diverse cellular origins might be temporally activated during the S phase of the eukaryotic cell cycle.

Animals↗
PubMed1996

Genetic analysis of the activation domain of bovine papillomavirus protein E2: its role in transcription and replication.

The bovine papillomavirus protein E2 serves dual functions in viral transcription and in the initiation of viral replication. As a transcription factor, E2 can cooperatively interact with cellular proteins such as SP1 and stimulate transcription of distal promoters. In replication, E2 and the helicase El are the only viral proteins required for accurate replication of templates containing the viral origin. The amino terminus of E2 is a functionally separable domain critical for activation of both replication and transcription; its primary sequence is conserved between many strains of papillomavirus. We targeted conserved residues spanning the activation domain and constructed a series of 30 amino acid substitution mutants. These mutant E2 genes were analyzed for the ability to activate DNA replication and gene expression in cells. The majority of the substitutions affected the ability of E2 to support both viral replication and transcriptional activation, revealing substantial overlap of the functional determinants for these two processes. Replication and transcription activities are genetically separable, however, as mutations at amino acids 73 and 74 retained replication function but failed to activate transcription. Additionally, a mutation at position 39 substantially reduced replication activity but left transcriptional activation intact. Interestingly, over two-thirds of the mutations analyzed reduced function and protein accumulation, many in a temperature-dependent manner. The correspondence between the replication and transcription phenotypes of mutations spanning the activation domain may indicate that the entire region is folded into a single domain required for both functions.

Amino Acid Sequence↗
PubMed1997

Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication approximately 2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced approximately 5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (approximately 2-fold) on oriS function. Although the cis-acting elements that mediate the NGF- and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.

Animals↗
PubMed1998

Rescue and autonomous replication of adeno-associated virus type 2 genomes containing Rep-binding site mutations in the viral p5 promoter.

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.

DNA Helicases↗
PubMed1999

Induction and maintenance of autonomous flock house virus RNA1 replication.

The nodavirus flock house virus (FHV) has a bipartite, positive-sense, RNA genome that encodes the catalytic subunit of the RNA replicase and the viral capsid protein precursor on separate genomic segments (RNA1 and RNA2, respectively). RNA1 can replicate autonomously when transfected into permissive cells, allowing study of the kinetics of RNA1 replication in the absence of either RNA2 or capsid proteins. However, RNA1 replication ceases ca. 3 days after transfection despite the presence of replication-competent RNA. We examined this inhibition by inducing the expression of RNA1 in cells from a cDNA copy that was under the control of a hormone-regulated RNA polymerase II promoter. This system reproduced the shutoff of RNA replication when DNA-templated primary transcription was turned off. Continued primary transcription partially alleviated the shutoff and maintained the rate of RNA replication for several days at a steady-state level approximately one-third that of the peak rate. After shutoff, RNA replication could be restored by transferring the resulting intracellular RNA to fresh cells or by reinducing primary transcription, indicating that cessation of replication occurred despite the competence of both the viral RNA and the cytoplasmic environment. These data suggest that there is a mechanism by which replication is shut off at late times after transfection, which may reflect the natural endpoint of the replicative cycle.

Animals↗
PubMed1999

Replication of Aleutian mink disease parvovirus in vivo is influenced by residues in the VP2 protein.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.

Aleutian Mink Disease↗