Adenovirus recombination in normal and repair-deficient human fibroblasts

The ability of adenovirus to recombine in fibroblasts derived from several of the human cancer-prone syndromes has been examined. Cells deficient in uv-repair, namelyXeroderma pigmentosum (XP) and XP variant, are recombinant-proficient and yield 10³–10⁴ infectious virus per infected cell, as do the control fibroblasts, GM17. Similarly, Fanconi's anemia fibroblasts which are sensitive to DNA crosslinking agents and display elevated levels of karyological changes are recombination-proficient. Bloom's syndrome fibroblasts on the other hand, generally yield lower quantities of virus but the adenovirus recombination frequency is elevated some twofold, compared with values obtained in parallel experiments with GM17. The DNA structures of heterotypic Ad5 × Ad2ND1 recombinants obtained from Bloom's and from GM17 fibroblasts are similar, suggesting that the adenovirus recombination mechanism(s) in the two cell types are also similar.     

Replication and interaction of virus DNA and cellular DNA in mouse cells infected by a human adenovirus

C57 black mouse cells infected with human adenovirus type 5 (Ad5) produced large amounts of early virus proteins, small amounts of late virus proteins and less than 0.2 infectious units (i.u.)/cell of infectious virus. Many cells died but the cultures recovered. Virus DNA and cellular DNA were synthesized. Some Ad5 DNA sedimented with cell DNA in alkaline sucrose, but virus DNA was rapidly lost from the culture after recovery and none of 28 unselected cloned survivors contained detectable amounts of virus DNA or antigens. Ad5 ts36 was temperature-sensitive for virus DNA replication in mouse cells, but ts125 was detective at 32.5 degrees C as well as at 39.9 degrees C. No difference was detected in the percentage of virus DNA that sedimented in alkali with cell DNA, in mouse cells infected by Ad5 ts+, ts36 or ts125 at 32.5 or 39.9 degrees C. All parts of the virus genome were equally represented in virus DNA that sedimented with cell DNA, in mouse cells infected by Ad5 ts+ or ts36 at either temperature.     

乙型肝炎病毒表面抗原S基因在人5型重组腺病毒中的表达及其特性

The hepatitis B virus (HBV) S gene, PreS2 + S genes and the late phase expression cassette (MTI) + HBV S genes were separately cloned into Ad5 vector downstream of E3 promoter (pAd5 deltaE3 provided by Wyeth Co.). The above constructed plasmids and Ad5 DNA EcoR I A fragment were cotransfected into 293 cells. The progeny adenoviruses named rAd5S, rAd5MS, rAd5S2S were harvested for analysis. The recombinants were isolated and analyzed by PCR, using two primers specific to the HBV S genes. The expressed products were detected by ELISA and RIA. The recombinant containing MIT + HBV S genes (rAd5MS) was identified to be ELISA positive, whereas the other two recombinants (rAd5S, rAd5S2S) were negative to ELISA, but positive to RIA. The results indicated that adenovirus E3 early promoter could express the inserted foreign genes, and MIT worked well in the E3 region of Ad5 and could increase the expression capacity of the recombinants. The conditions for foreign gene expression and genetic stability of the recombinant viruses were studied in detail. There was no wild Ad5 discovered during the cotransfection experiments. The present study provides some experiences for studying adenovirus recombination.     

Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Successful oral rabies vaccination of raccoons with raccoon poxvirus recombinants expressing rabies virus glycoprotein

Two infectious raccoon poxvirus (RCN) recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants. Expression of G protein was controlled by vaccinia virus promoter P7.5 (early/late class) or by P11 (late class). Immunoprecipitation of infected cell extracts indicated that both of the RCN recombinants directed faithful expression of G protein. Raccoons that were fed polyurethane baits loaded with either recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with lethal raccoon rabies street virus.     

Recombination between simian virus 40 and adeno-associated virus: virion coinfection compared to DNA cotransfection

Recombination between simian virus 40 (SV40) and adeno-associated virus (AAV) has been detected, by infectious center in situ plaque hybridization procedures, after both DNA contransfection and virion coinfection of monkey BSC-1 cells. The number of cells producing recombinants (1 in a 1000) was the same irrespective of the way in which the SV40 and AAV genomes were delivered to the cell, despite the fact that 5-10 times more cells were infected after virion coinfection. Several other dosage-response parameters of the recombination process consequent to virion coinfection were comparable to those after DNA cotransfection. The sole difference observed between the two infection systems was that the SV40/AAV recombinants formed after virion coinfection contained an inordinately high proportion of AAV terminal DNA sequences. By separating the SV40 and AAV infections in time, such that the AAV infection was delayed until after certain events in the SV40 cycle had taken place, an optimum phase for recombination in the SV40 cycle was identified. This phase occurs a few hours after infection, well before the onset of SV40 DNA replication and the synthesis of SV40-specific early proteins.     

Replicating Adenovirus-Simian Immunodeficiency Virus (SIV) Vectors Efficiently Prime SIV-Specific Systemic and Mucosal Immune Responses by Targeting Myeloid Dendritic Cells and Persisting in Rectal Macrophages,Regardless of Immunization Route

Although priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, followed by HIV/SIV envelope boosting, has proven highly immunogenic, resulting in protection from SIV/simian-human immunodeficiency virus (SHIV) challenges, Ad5hr recombinant distribution, replication, and persistence have not been examined comprehensively in nonhuman primates. We utilized Ad5hr-green fluorescent protein and Ad5hr-SIV recombinants to track biodistribution and immunogenicity following mucosal priming of rhesus macaques by the intranasal/intratracheal, sublingual, vaginal, or rectal route. Ad recombinants administered by all routes initially targeted macrophages in bronchoalveolar lavage (BAL) fluid and rectal tissue, later extending to myeloid dendritic cells in BAL fluid with persistent expression in rectal mucosa 25 weeks after the last Ad immunization. Comparable SIV-specific immunity, including cellular responses, serum binding antibody, and mucosal secretory IgA, was elicited among all groups. The ability of the vector to replicate in multiple mucosal sites irrespective of delivery route, together with the targeting of macrophages and professional antigen-presenting cells, which provide potent immunogenicity at localized sites of virus entry, warrants continued use of replicating Ad vectors.     

Adenovirus early region 1b gene function required for rescue of latent adeno-associated virus

Various early and late temperature-sensitive mutants of adenovirus (Ad) were able to support adeno-associated virus (AAV) DNA replication in either coinfections or in the rescue of AAV from latently infected human (Detroit 6) cells. Group I Ad host range mutants (hr 3, early region 1a) were unable to help AAV DNA replication in either case. Group II Ad host range mutants (hr 6, early region 1b), however, were able to help AAV DNA replication in coinfection but were unable to rescue AAV DNA replication in latently infected Detroit 6 cells. Thus, an Ad function(s), in addition to those required for AAV DNA replication, is needed for AAV rescue.     

Restricted replication of mouse adenovirus strain FL in mouse Balb/c cells transformed by Simian Adenovirus 7

Mouse kidney cells (Balb/c cells), in which mouse adenovirus strain FL (Ad FL) replicates, become restricted in their capacity to support Ad FL growth after transformation by simian adenovirus 7 (SA7). In transformed cells (Balb/c SA7 cells), the duration of the Ad FL cycle is significantly longer than in normal cells; some virus DNA replication and virus protein synthesis occur but the yield of infectious Ad FL particles is greatly reduced. Only a few cells produce virus as estimated by the number of infectious centers. However, no significant differences are noted in the adsorption and in the penetration of Ad FL in both cell types. Thus, the restrictive event in virus multiplication in Balb/c SA7 cells occurs probably after viral uncoating and before viral DNA synthesis.     

Recombination in alphaherpesviruses

Within the Herpesviridae family, Alphaherpesvirinae is an extensive subfamily which contains numerous mammalian and avian viruses. Given the low rate of herpesvirus nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in alphaherpesviruses is intimately linked to DNA replication. Both viral and cellular proteins participate in this recombination-dependent replication. The presence of inverted repeats in the alphaherpesvirus genomes allows segment inversion as a consequence of specific recombination between repeated sequences during DNA replication. High molecular weight intermediates of replication, called concatemers, are the site of early recombination events. The analysis of concatemers from cells coinfected by two distinguishable alphaherpesviruses provides an efficient tool to study recombination without the bias introduced by invisible or non-viable recombinants, and by dominance of a virus over recombinants.Intraspecific recombination frequently occurs between strains of the same alphaherpesvirus species. Interspecific recombination depends on enough sequence similarity to enable recombination between distinct alphaherpesvirus species. The most important prerequisite for successful recombination is coinfection of the individual host by different virus strains or species. Consequently the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology, virulence and latency. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.     

Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase

The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad pol). Ad pol and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad pol a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad pol genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.     

Differential complementation of adenovirus type 5 temperature-sensitive early mutants by adenovirus types 3 and 12

We have assayed the ability of human adenoviruses from heterologous subgroups to complement early temperature-sensitive mutants of the C group virus Ad5 by analyzing coinfections at the restrictive temperature for serotype-specific DNA and late protein synthesis. Our results indicate that the B group virus AM, does not complement either ts125 or the N complementation group of Ad5 (ts36, ts69, and ts149). In contrast, studies with Ad12, an A group virus, and these mutants, indicate a differential complementation in that Ad12 complements all of the mutants in the Ad5 N complementation group but does not complement ts125. Failure to complement the ts125 defect in coinfected cells has been shown to be due to the inability of the heterologous wt gene product to substitute for the ts125 gene product directly at the level of DNA replication and not at some earlier event in the virus growth cycle.     

Molecular biology of adenovirus type 2 semipermissive infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection

As an initial step toward understanding the mechanisms underlying host cell restriction of adenovirus 2 (Ad2) replication, we have studied various cell lines derived from hamster (CHO-K1), rat (CREF, NRK-49F, C-3, C-9), and mouse (3T3-Swiss) tissues to determine their degree of permissivity to Ad2 replication. For each cell line tested, the time course of Ad2 growth was determined; the yield of infectious virus, as measured by titration on HeLa cell monolayers, was reduced 3 to 5 logs. This result is independent of the multiplicity of infection at multiplicities between 4 and 100 plaque-forming units (PFU) per cell. The Western immunoblotting technique was used to quantitate the amounts of early proteins (E1A 45-54K proteins, E1B 21 and 58K proteins, E2A 72K DNA binding protein) and late structural proteins (hexon, fiber) produced during restricted infections. All cell lines expressed 72K DNA binding protein and variable levels of other early proteins. C-3, C-9, and NRK-49F cells expressed hexon as well as low, but detectable levels of fiber protein. Mouse 3T3-Swiss cells failed to synthesize any detectable levels of late structural proteins. DNA synthesis analysis indicated all rodent cell lines were capable of replicating viral DNA. A decreased rate of viral DNA synthesis was observed in CREF cells. Evidence is presented which suggests newly synthesized viral DNA is unstable in 3T3-Swiss cells.     

Replication of an adenovirus type 34 mutant DNA containing tandem reiterations of the inverted terminal repeat

A mutant of human adenovirus type 34 (Ad34) has been isolated which contains DNA molecules with tandem reiterations of from two to eight copies of a 131-bp sequence within the right-sided inverted terminal repetition. Terminal heterogeneity was not eliminated by repeated plaque purifications indicating that the population of DNA molecules with various numbers of reiterations could rapidly evolve from the DNA of a single virus particle. These enlarged DNA molecules were capable of replication both in vivo and in vitro. The nucleotide sequence of the mutant Ad34 inverted terminal repetitions contained most of the essential features of the Ad origin of DNA replication. These features include the ATAATATACC sequence which is present between the highly conserved bases 9-18 in all human adenoviruses, as well as the consensus sequences for the binding of nuclear factor I and nuclear factor III. However, the reiterated sequences lacked a dG appropriately placed on the template strand to serve as a potential site for internal initiation. It appears that the rapid amplification of two to eight copies of the reiterated terminal sequences does not arise from internal initiation during replication but probably from homologous recombination.     

Integrative recombination between adenovirus type 12 DNA and mammalian DNA in a cell-free system: joining by short sequence homologies

A cell-free system was developed to investigate the mechanism of how junctions are formed between viral and cellular DNAs during adenoviral DNA integration into the hamster cell genome. Recombination between the segment of adenovirus type 12 (Ad12) DNA, that comprises sequence coordinates 20 885–24 053, subsequently termed PstI-D fragment and the hamster preinsertion DNA sequence p7 was studied in a cell-free system. The p7 DNA segment had served as viral DNA integration site in the Ad12-induced tumor CLAC1. Nuclear extracts initially from uninfected BHK21 hamster cells were fractionated by a series of chromatographic steps. DNAs of the in vitro generated recombinants were analyzed in detail. In the course of the recombination reaction, the two linear molecules were joined. The reaction took place between two short homologous sequences one of which was always at or very close to a DNA terminus, the second one could be several kilobase pairs remote from a DNA terminus. Apparently, the nucleotide sequence at the terminus of one recombining molecule determined the point of junction by searching for short homologies in the partner molecule. The recombination reaction was not conservative, the sequences in-between the short sequence homologies and one of the short sequence homologies were deleted in the in vitro recombinants. Two main criteria influenced the choice of interacting short sequence homologies: perfect homologies of 8–9 bp were most frequently found, they were preferred over more extended, but less perfect homologies. Comparing different short sequence homologies with similar stabilities, those combinations seemed to be chosen in the reaction which led to a minimal loss of nucleotides in the recombinants. The in vitro activity was found in nuclear extracts from both hamster and human cells. The activity was, hence, available for Ad12 DNA in productively infected human and abortively infected hamster cells. The specific recombination activity was increased in nuclear extracts of hamster cells abortively infected with Ad12. The junction sites in the recombinants, which were generated by the cell-free system, were very similar to junctions between adenoviral and cellular DNAs cloned from Ad12-induced tumor cells and Ad12-transformed cell lines.     

Multiple recombination sites at the 5'-end of murine coronavirus RNA

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.     

Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination.