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PubMed2006

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Animals↗
PubMed2025

Probing orthobunyavirus reassortment using Bunyamwera and Batai viruses as models.

Reassortment is a critical evolutionary mechanism for segmented viruses, enabling the exchange of intact genome segments during co-infection and driving orthobunyavirus evolution; however, the molecular mechanisms underpinning this process remain unclear. With over 100 orthobunyavirus species, many of which are significant human and veterinary pathogens, understanding how reassortment influences transmissibility and virulence is essential for preempting the emergence of novel pathogens. Here, we use Bunyamwera virus (BUNV) and Batai virus (BATV) as models to explore orthobunyavirus reassortment through reverse genetics. We established the first reverse genetics system for BATV, generated reassortants, and employed minigenome assays to assess replication machinery compatibility. Additionally, we developed a novel hybridization chain reaction assay for high-resolution visualization of viral RNA segments. Our findings revealed that all six reassortants between BUNV and BATV are viable, exhibiting notable phenotypic differences in interferon-deficient (IFNAR-/-) mice. This work introduces essential tools and new insights into orthobunyavirus reassortment and pathogenesis, laying the groundwork for understanding this critical evolutionary process.

Animals↗
PubMed1998

Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes.

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.

Animals↗
PubMed2005

Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs.

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.

Animals↗
PubMed2006

Identification of 5' and 3' cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5' AU-rich sequences restored replication of a 5'-proximal 7-nucleotide deletion mutant.

We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had specific infectivities of >5 x 10(5) PFU/microg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5' ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5' sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3' cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.

3' Flanking Region↗
PubMed2001

Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus.

A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.

Animals↗
PubMed2006

Helper virus-independent trans-replication of hepatitis C virus-derived minigenome.

We have previously described a synthetic T7-driven cDNA minigenome containing the antisense sequence of luciferase gene and internal ribosome entry site of encephalomyocarditis virus flanked by 5'- and 3'-end sequences of hepatitis C virus (HCV) that contain cis-acting replication elements. Synthesis of minus-strand RNA from the artificial minigenome was determined by using Huh-7 cells harboring autonomously replicating HCV subgenome as a helper for provision of functional replication components. To further confirm and extend these studies, we investigated here whether the minigenome replication system could be reconstituted by transfection of naïve Huh-7 cells with plasmid expressing nonstructural (NS) proteins. Reporter assay and Northern blot analysis revealed that trans-expression of NS proteins from 3 to 5 resulted in high level of luciferase activity and synthesized minus-strand RNA. The analogous result was also obtained with the minigenome derived from HCV 2a, and both HCV 1b- and 2a-derived NS protein were able to support the chimeric minigenomes whose 5'- or 3'-end was replaced by the respective region of the heterologous virus. These results provide a basis for establishing the reverse genetic system that is helpful to study cis- and trans-acting factors involved in HCV RNA replication.

Cell Line, Tumor↗
PubMed2005

Replication strategies of rabies virus.

Rabies virus (RV) is a prototype neurotropic virus that causes fatal disease in human and animals. RV infects hosts at the periphery, enters motoneurons or sensory nerves and moves to the central nervous system (CNS) via retrograde axonal transport. At later stages, there is also centrifugal spread to major exit portals, such as the salivary glands. Transmission to other hosts is facilitated by behavioral changes related to the CNS infection. Successful accomplishment of the RV infectious cycle depends on multiple functions of the virus, and of individual virus proteins, all together defining the typical pathogenicity and virulence, i.e. the biological fitness of this virus. In particular, it appears important for RV to sneak into the host without causing pronounced host responses and to preserve, at least for some time, the integrity of infected cells and of the neuronal network. The availability of reverse genetics systems that allow generation of engineered recombinant RV has provided tools for a more detailed analysis of viral functions relevant to the typical RV pathogenesis. Novel developments such as tracking of live fluorescent RV are further increasing the opportunities to decipher RV pathogenicity factors. In this review, we describe different aspects of the molecular biology of RV that are relevant to pathogenesis, with a particular emphasis on the accurate control of RV transcription, gene expression, and replication. In addition, the role of individual virus proteins in maintaining host cell integrity and supporting retrograde transport is discussed. The potential of recombinant RVs with single or multiple pathogenicity factors eliminated is being discussed in terms of vaccine and virus vector development.

Amino Acid Sequence↗
PubMed2005

Novel antiviral strategies to combat human Arenavirus infections.

Arenaviruses merit significant attention both as tractable model systems to study acute and persistent viral infections, and as clinically important human pathogens. Evidence indicates that LCMV remains present in the USA and Europe and capable of causing significant morbidity in infected individuals, likely being a neglected human pathogen. Moreover, new arenaviruses are being discovered in the Americas on the average of one every three years, with some of them causing severe hemorrhagic fever. In addition, weaponized forms of these viruses pose a real threat as agents of bioterrorism. Therefore, it is important to develop effective vaccines and better antiviral drugs to combat the dual threats of naturally occurring and intentionally introduced Arenavirus infections. The development of arenavirus reverse genetic systems is allowing investigators to conduct a detailed molecular characterization of the viral cis-acting signals and trans-acting factors that control each of the steps of the Arenavirus life cycle, including RNA synthesis, packaging and budding. We will discuss how this new knowledge is facilitating the establishment of novel assays to identify and characterize compounds capable of interfering with specific steps of the virus life cycle. Likewise, the ability to generate predetermined specific mutations within the arenavirus genome, and analyze their phenotypic expression, would significantly contribute to the elucidation of arenavirus-host interactions, including the bases of their ability to persist, as well as to cause severe HF (hemorrhagic fever) disease in humans. These approaches could also lead to the development of novel potent and safe Arenavirus vaccines.

Antibodies, Viral↗
PubMed2005

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations.

OBJECTIVES AND METHODS: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. RESULTS AND CONCLUSIONS: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.

Animals↗
PubMed2006

Rescue of the prototypic Arenavirus LCMV entirely from plasmid.

We document a helper-independent reverse genetics system for rescuing infectious arenaviruses from cloned cDNAs. We constructed plasmids containing full-length cDNAs of the antigenomic (ag) L and S segments of the Armstrong (ARM) strain of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) flanked at their 5'- and 3'-termini by the T7 RNA polymerase (T7RP) promoter and ribozyme sequences, respectively. These plasmids directed intracellular synthesis of viral L and S ag RNA species in cells expressing plasmid-supplied T7RP. Co-expression of plasmid-supplied LCMV trans-acting factors, nucleoprotein (NP) and polymerase (L), resulted in replication and expression of L and S ag and genome RNA species, and generation of LCMV infectious progeny termed rT7/LCMV. The recombinant rT7/LCMV was unequivocally identified based on a genetic tag introduced in the recombinant S segment. In addition, rT7/LCMV exhibited growth and biological properties predicted for an ARM-like LCMV. To our knowledge, this is the first documented Arenavirus rescue, as well as of an ambisense negative strand (NS) RNA virus, entirely from cloned cDNAs. Our results extend the use of reverse genetic approaches for DNA-mediated virus rescue to all known virus families with NS RNA genome.

Animals↗
PubMed2005

Genome trimming: a unique strategy for replication control employed by Borna disease virus.

Genome and antigenome synthesis of negative-strand RNA viruses is initiated at promoters located in inverted terminal repeats (ITR). The ITR of Borna disease virus (BDV), a persisting neurotropic virus with a nuclear replication phase, are exceptional in that they appear to be noncomplete. Our analysis showed that the vast majority of genomic and antigenomic RNA molecules of BDV lack four 5'-terminal nucleotides required for perfect complementarity with the 3' ITR. By using a previously undescribed reverse genetics system, we investigated whether the structure of the ITR would affect virus propagation. BDV rescued from cDNA encoding complete ITR (rBDVc) showed wild-type virulence, whereas virus rescued from cDNA encoding a viral genome with noncomplete ITR (rBDVnc) was strongly attenuated. Both recombinant viruses expressed similar RNA and protein levels in persistently infected cells. However, rBDVnc particles were less infectious, indicating that complete ITR are required for high viral replicase but not transcriptase activity. Interestingly, genomic RNA from purified rBDVc particles lacked 5'-terminal nucleotides like authentic BDV, strongly suggesting programmed genome truncation. By specifically trimming its genome at the 5' terminus, BDV seems to limit viral genome amplification, which may favor noncytolytic viral persistence.

Base Sequence↗
PubMed2004

The NV gene of snakehead rhabdovirus (SHRV) is not required for pathogenesis, and a heterologous glycoprotein can be incorporated into the SHRV envelope.

Snakehead rhabdovirus (SHRV) affects warm-water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its "nonvirion" (NV) gene. To examine the function of the NV gene, we used a recently developed reverse genetic system to produce a viable recombinant SHRV carrying an NV gene deletion. The recombinant virus was produced at the same rate and same final concentrations as the wild-type virus in cultured fish cells in spite of the NV gene deletion. The role of the NV protein in fish pathogenesis was also investigated. Zebra fish (Danio rerio) were infected with the NV deletion mutant or with a recombinant virus containing a copy of the SHRV genome, and similar mortality rates as well as final mortalities were recorded, suggesting no apparent role for the NV protein in fish pathogenesis. Interestingly, the unsuccessful rescue of fully viable recombinants with genomes containing deletions in the G/NV gene junction suggested a role for the gene junction in virus transcription and replication. Finally, we demonstrated that the SHRV glycoprotein can be replaced by the glycoprotein of infectious hematopoietic necrosis virus (IHNV) or by a hybrid protein composed of SHRV and IHNV sequences.

Animals↗
PubMed1998

Generation of a mutant infectious bursal disease virus that does not cause bursal lesions.

A reverse genetics system for birnavirus, based on synthetic transcripts of the infectious bursal disease virus (IBDV) genome, was recently developed (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). To study the function of the 17-kDa nonstructural (NS) protein in viral growth and pathogenesis, we constructed a cDNA clone of IBDV segment A in which the first and only initiation codon (ATG) of NS protein was mutated to a stop codon (TAG). Transfection of Vero cells with combined transcripts of either modified or unmodified segment A, and with segment B, generated viable IBDV progeny. When chicken embryo fibroblast cells infected with transfectant viruses were analyzed by immunofluorescence assays using NS-specific antiserum, the mutant virus did not yield a fluorescence signal, indicating a lack of NS protein expression. Furthermore, replication kinetics and cytotoxic effects of the mutant virus were compared with those of the parental attenuated vaccine strain of IBDV (D78) in vitro. The mutant virus grew to slightly lower titers than D78 virus and exhibited decreased cytotoxic and apoptotic effects in cell culture. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78 or mutant virus and analyzed their bursa for histopathological lesions. The recovered D78 virus caused microscopic lesions and atrophy of the bursa, while the mutant virus failed to induce any pathological lesions or clinical signs of disease. In both instances, the virus was recovered from the bursa, and the presence or absence of mutation in these viruses was confirmed by nucleotide sequence analysis of NS gene. Although the mutant virus exhibited a delay in replication in vivo, it induced levels of IBDV neutralizing antibodies that were similar to those of D78 virus. In addition, no reversion of mutation was detected in the mutant virus recovered from inoculated chickens. These results demonstrate that NS protein is dispensable for viral replication in vitro and in vivo and that it plays an important role in viral pathogenesis. Thus, generation of such NS protein-deficient virus will facilitate the study of immunosuppression and aid in the development of live-attenuated vaccines for IBDV.

Animals↗
PubMed2000

Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites.

The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3' polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT-PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs. The results showed that although the SV5 NP, M, and SH genes encode U tracts of seven, four, and six U residues, respectively, their mRNAs contain similar polyA tails of approximately 250-290 bases. These results indicate that the variable gene end U tracts are functionally equivalent in directing polyadenylation. A reverse genetics system based on a dicistronic minigenome containing the SH-HN gene junction was used to test the hypothesis that the variable U tracks affect the efficiency of transcription termination. Minigenome templates containing an SH gene end with a long U tract of six residues (U6) directed efficient transcription termination and reinitiation at the downstream HN start site with no nucleotide preference for the downstream intergenic region. Surprisingly, truncating the SH gene end U tract to four residues (U4) did not affect SH termination but, rather, reduced downstream HN reinitiation to 20-30% of wild-type levels. Efficient HN reinitiation could be restored to mutant U4 templates in either of two ways: by increasing the U-tract length from four to six residues or by increasing the length of the intergenic region. Efficient HN reinitiation required a minimum of six bases between the last nucleotide in SH and the first nucleotide in HN. We propose that for some paramyxoviruses, the gene end U tract serves a previously unrecognized role as a spacer region between the gene end and gene start sites.

Animals↗
PubMed1996

Nonconserved nucleotides at the 3' and 5' ends of an influenza A virus RNA play an important role in viral RNA replication.

The genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. The signals required for replication, transcription, and packaging of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form "panhandle" or "fork" structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. In contrast, the nonconserved noncoding nucleotides located close to the open reading frame of the viral RNAs had not been implicated in RNA transcription. Using a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these nonconserved noncoding regions. Deletion either of the nucleotide residues between the poly(U) stretch and the stop codon at the 5' end or of the nucleotides between position 15 and the start codon at the 3' end did not affect the amount of NA-RNA species found in virions or infected cells. However, a combination of deletions at both the 3' and the 5' ends decreased by 60 times the levels of NA-specific viral RNA found in infected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady-state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of infuenza A/WSN/33 virus also showed a reduction in the NA-specific viral RNA in virions and in infected cells. The present results demonstrate that the nonconserved nucleotides at the 3' and 5' ends of the NA-RNA segment of influenza A virus play an important role in the replication of this segment.

Animals↗
PubMed1996

The nonstructural C protein is not essential for multiplication of Edmonston B strain measles virus in cultured cells.

Measles virus (MV) is a highly contagious agent which causes a major health problem in developing countries. Efficacious and safe live attenuated vaccine strains are available, but for the elimination of measles a better knowledge about the molecular biology of MV appears crucial. Whereas the roles of the six structural proteins in the replication cycle are known, the functions of the two nonstructural proteins C and V are unclear, which is also true for related viruses. In vitro studies implicating Sendai virus suggest that the C protein might be involved in downregulating viral mRNA synthesis (J. Curran, J.B. Marq, and D. Kolakofsky, Virology 189, 647-656, 1992). However, not all members of the Paramyxovirinae subfamily encode this protein, raising the question about its importance for the viral replication cycle. Taking advantage of a recently developed reverse genetics system allowing MV recovery from cloned DNA (F. Radecke, P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, C. Dötsch, G. Christiansen, and M.A. Billeter, EMBO J. 14, 5773-5784, 1995), the question was addressed whether the C protein is essential for the life cycle of MV. A plasmid was constructed to produce a derivative of the Edmonston B vaccine strain, MV C- EdB, having its C reading frame silenced by two point mutations. The C- mutant MV could indeed be rescued, and it multiplies in cultured cells without obvious impairment.

Animals↗
PubMed1996

The efficiency of Sendai virus genome replication: the importance of the RNA primary sequence independent of terminal complementarity.

From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3' end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3'/5' end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identify cis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1-31 of the antigenome 3' end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing.

Animals↗