Philomena Ostapchuk

The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome

ABSTRACT Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a cis-acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5′ TTTG-N8-CG 3′. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral genome did not reduce viral gene expression or viral DNA replication but eliminated the production of infectious virus. We suggest that the L4 22-kDa protein, in conjunction with IVa2, plays a critical role in the recognition of the packaging domain of the Ad genome that leads to viral DNA encapsidation. The L4 22-kDa protein is also involved in recognition of transcription elements of the Ad major late promoter.

Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences

ABSTRACT Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.

Pseudopackaging of Adenovirus Type 5 Genomes into Capsids Containing the Hexon Proteins of Adenovirus Serotypes B, D, or E

ABSTRACT Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.

Adenovirus IVa2 Protein Binds ATP

ABSTRACT IVa2 is an essential, multifunctional protein of adenovirus (Ad) supporting packaging of the viral genome into the capsid, assisting in assembly of the capsid, and activating Ad late transcription. A comparison of IVa2 protein sequences from different species of Adenoviridae shows conserved motifs associated with binding and hydrolysis of ATP (Walker A and B motifs). ATPases are essential proteins of bacteriophage packaging motors, and such activity may be required for Ad packaging. Results presented here show that the Ad2 IVa2 protein binds ATP in vitro and that sequences in the Walker A and B motifs are necessary for this activity.

Binding of CCAAT Displacement Protein CDP to Adenovirus Packaging Sequences

ABSTRACT Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.

Identification of a second cryptic lambdoid prophage locus in the E. coli K12 chromosome

SummaryIn addition to the cryptic lambdoid prophage genes that are known to reside at the rac locus in Escherichia coli K12 strains, a second cryptic lambdoid prophage has been located near the gal operon. This prophage was shown to contain DNA that is homologous to the QSR genes of λ phage.

Methylation-dependent and -independent DNA binding of nuclear factor EF-C

Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus enhancer regions. In this paper, we have characterized new and divergent EF-C binding sites in several viral regulatory regions. We also have demonstrated that EF-C binds to certain DNA sites only when CpG dinucleotide base pairs are methylated (m5C). EF-C binds to other sites in a methylation-independent manner. Based on similar binding properties and identical binding sites, it is very likely that EF-C corresponds to the nuclear protein MDBP previously identified by virtue of binding to methylated DNA.

Conserved genes in enteric bacteria are not identical.

SummaryIn addition to ribosomal RNA genes, a relatively small number of genes are highly conserved between Escherichia coli andSalmonella typhimurium on the one hand, and either Serratia marcescens or Proteus morganii on the other hand. The conserved non-rRNA genes are not the same in S. marcescens and P. morganii. Different genetic segments have maintained nucleotide sequence similarity to parts of the E. coli and S. typhimurium genomes in the course of the evolution of S. marcescens and P. morganii.