IVa2 Protein Research Articles

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24–48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1–25 and 373–448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4–18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1–25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1–25 and 120–140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Formation of adenovirus DNA replication compartments and viral DNA accumulation sites by host chromatin regulatory proteins including NPM1.

The adenovirus (Ad) genome is believed to be packaged into the virion by forming a chromatin-like structure. The replicated viral genome is likely to be condensed through binding with viral core proteins before encapsidation. Replicated viral genomes accumulate in the central region of the nucleus, which we termed virus-induced postreplication (ViPR) body. However, the molecular mechanism by which the nuclear structure is reorganized and its functional significance in virus production are currently not understood. In this study, we found that viral packaging protein IVa2, but not capsid proteins, accumulated in the ViPR body. In addition, nucleolar chromatin regulatory proteins, nucleophosmin 1 (NPM1), upstream binding factor, and nucleolin accumulated in the ViPR body in late-stage Ad infection. NPM1 depletion increased the nuclease-resistant viral genome and delayed the ViPR body formation. These results suggested that structural changes in the infected cell nucleus depend on the formation of viral chromatin by host chromatin regulatory proteins. Because NPM1 depletion decreases production of the infectious virion, we propose that host factor-mediated viral chromatin remodeling and concomitant ViPR body formation are prerequisites for efficient encapsidation of Ad chromatin.

Adenoviruses of the most ancient primate lineages support the theory on virus-host co-evolution.

The scarcity or complete lack of information on the adenoviruses (AdVs) occurring in the most ancient non-human primates resulted in the initiation of a study for exploring their abundance and diversity in prosimians and New World monkeys (NWMs). In order to assess the variability of these AdVs and the possible signs of the hypothesised virus-host co-evolution, samples from almost every family of NWMs and prosimians were screened for the presence of AdVs. A PCRscreening of 171 faecal or organ samples from live or dead, captive or wild-living prosimians and NWMs was performed. The PCR products from the gene of the IVa2 protein were sequenced and used in phylogeny calculations. The presence of 10 and 15 new AdVs in seven and ten different species of prosimians and NWMs was revealed, respectively. Phylogenetic analysis indicated that the tentative novel AdVs cluster into two separate groups, which form the most basal branches among the primate AdVs, and therefore support the theory on the co-evolution of primate AdVs with their hosts. This is the first report that provides a comprehensive overview of the AdVs occurring in prosimians and NWMs, and the first insight into the evolutionary relationships among AdVs from all major primate groups.

Adenoviral E4 34K protein interacts with virus packaging components and may serve as the putative portal

Studies on dsDNA bacteriophages have revealed that a DNA packaging complex assembles at a special vertex called the ‘portal vertex’ and consists of a portal, a DNA packaging ATPase and other components. AdV protein IVa2 is presumed to function as a DNA packaging ATPase. However, a protein that functions as a portal is not yet identified in AdVs. To identify the AdV portal, we performed secondary structure analysis on a set of AdV proteins and compared them with the clip region of the portal proteins of bacteriophages phi29, SPP1 and T4. Our analysis revealed that the E4 34K protein of HAdV-C5 contains a region of strong similarity with the clip region of the known portal proteins. E4 34K was found to be present in empty as well as mature AdV particles. In addition, E4 34K co-immunoprecipitates and colocalizes with AdV packaging proteins. Immunogold electron microscopy demonstrated that E4 34K is located at a single site on the virus surface. Finally, tertiary structure prediction of E4 34K and its comparison with that of single subunits of Phi29, SPP1 and T4 portal proteins revealed remarkable similarity. In conclusion, our results suggest that E4 34K is the putative AdV portal protein.

Tropism and infectivity of duck-derived egg drop syndrome virus in chickens.

Egg drop syndrome virus (EDSV) can markedly decrease egg production in laying hens. Duck is the natural host of EDSV. EDSV derived from ducks abrogate egg drop in laying hens. We have previously confirmed that duck-derived EDSVs have a variety of replication activities in chick embryo liver (CEL) cells. However, it is currently unclear whether duck-derived EDSV could display tropism and adaptation in laying hens. This study assessed whether duck-derived EDSV can adapt to laying hens, and estimated the inducing factors. Complete genome sequences of duck-derived EDSVs (D11-JW-012, D11-JW-017, and D11-JW-032 isolates) with various replication efficiency in CEL cells and C10-GY-001 isolate causing disease in laying hens were analyzed to find their differences. Phylogenetic analysis of complete genome sequence revealed that C10-GY-001, D11-JW-032, and strain 127 virus as vaccine were clustered into the same group, with D11-JW-012 and D11-JW-017 clustered in another group. Comparison between D11-JW-012 isolate that poorly replicated and D11-JW-017 isolate that replicated well in CEL cells in same cluster revealed six amino acid differences on IVa2, DNA polymerase, endopeptidase, and DNA-binding protein. These amino acids might be key candidates enhancing cellular tropism in chicken. When the pathogenicities of these isolates in laying hens were compared, D11-JW-032 showed severe signs similar to 127 virus, D11-JW-017 showed intermediate signs, while D11-JW-012 showed almost no sign. Eleven amino acids differed between D11-JW-032 and D11-JW-017, and 17 amino acids were different between D11-JW-032 and D11-JW-012. These results suggest that EDSVs derived from ducks have various pathogenicities in laying hens. Key amino acid candidates might have altered their affinity to tropism of laying hens, causing difference pathogenicities.

Random sampling of the Central European bat fauna reveals the existence of numerous hitherto unknown adenoviruses.

From over 1250 extant species of the order Chiroptera, 25 and 28 are known to occur in Germany and Hungary, respectively. Close to 350 samples originating from 28 bat species (17 from Germany, 27 from Hungary) were screened for the presence of adenoviruses (AdVs) using a nested PCR that targets the DNA polymerase gene of AdVs. An additional PCR was designed and applied to amplify a fragment from the gene encoding the IVa2 protein of mastadenoviruses. All German samples originated from organs of bats found moribund or dead. The Hungarian samples were excrements collected from colonies of known bat species, throat or rectal swab samples, taken from live individuals that had been captured for faunistic surveys and migration studies, as well as internal organs of dead specimens. Overall, 51 samples (14.73%) were found positive. We detected 28 seemingly novel and six previously described bat AdVs by sequencing the PCR products. The positivity rate was the highest among the guano samples of bat colonies. In phylogeny reconstructions, the AdVs detected in bats clustered roughly, but not perfectly, according to the hosts' families (Vespertilionidae, Rhinolophidae, Hipposideridae, Phyllostomidae and Pteropodidae). In a few cases, identical sequences were derived from animals of closely related species. On the other hand, some bat species proved to harbour more than one type of AdV. The high prevalence of infection and the large number of chiropteran species worldwide make us hypothesise that hundreds of different yet unknown AdV types might circulate in bats.

Adenoviral L4 33K forms ring-like oligomers and stimulates ATPase activity of IVa2: implications in viral genome packaging.

The mechanism of genome packaging in adenoviruses (AdVs) is presumed to be similar to that of dsDNA viruses including herpesviruses and dsDNA phages. First, the empty capsids are assembled after which the viral genome is pushed through a unique vertex by a motor which consists of three minimal components: an ATPase, a small terminase and a portal. Various components of this motor exist as ring-like structures forming a central channel through which the DNA travels during packaging. In AdV, the IVa2 protein is believed to function as a packaging ATPase, however, the equivalents of the small terminase and the portal have not been identified in AdVs. IVa2 interacts with another viral protein late region 4 (L4) 33K which is important for genome packaging. Both IVa2 and 33K are expressed at high levels during the late stage of virus infection. The oligomeric state of IVa2 and 33K was analyzed in virus-infected cells, IVa2 and 33K transfected cells, AdV particles, or as recombinant purified proteins. Electron microscopy of the purified proteins showed ring-like oligomers for both proteins which is consistent with their putative roles as a part of the packaging motor. We found that the ATPase activity of IVa2 is stimulated in the presence of 33K and the AdV genome. Our results suggest that the 33K functions analogous to the small terminase proteins and so will be part of the packaging motor complex.

Analytical Ultracentrifugation as a Tool to Study Nonspecific Protein-DNA Interactions.

Analytical ultracentrifugation (AUC) is a powerful tool that can provide thermodynamic information on associating systems. Here, we discuss how to use the two fundamental AUC applications, sedimentation velocity (SV), and sedimentation equilibrium (SE), to study nonspecific protein-nucleic acid interactions, with a special emphasis on how to analyze the experimental data to extract thermodynamic information. We discuss three specific applications of this approach: (i) determination of nonspecific binding stoichiometry of E. coli integration host factor protein to dsDNA, (ii) characterization of nonspecific binding properties of Adenoviral IVa2 protein to dsDNA using SE-AUC, and (iii) analysis of the competition between specific and nonspecific DNA-binding interactions observed for E. coli integration host factor protein assembly on dsDNA. These approaches provide powerful tools that allow thermodynamic interrogation and thus a mechanistic understanding of how proteins bind nucleic acids by both specific and nonspecific interactions.

Characterization of the non-specific DNA binding properties of the Adenoviral IVa2 protein

Human Adenovirus (Ad) is a non-enveloped, icosahedral virus with a linear, double-stranded DNA genome. The Ad IVa2 protein is involved in multiple viral processes including viral late gene transcription and virus assembly. Previous studies have shown that IVa2 loads additional viral proteins onto conserved DNA elements within the Ad genome to regulate these viral processes. IVa2 also possesses strong non-specific DNA binding activity, and it is likely it uses this activity to recruit proteins to the conserved DNA elements. Here we have investigated the non-specific DNA binding activity of IVa2 using nitrocellulose/DEAE filter binding and sedimentation equilibrium techniques. We have analyzed our data using the McGhee and Von Hippel approach [1], and find that IVa2 binds with strong, positive nearest-neighbor cooperativity. In addition, we describe how to apply the McGhee and von Hippel approach to directly analyze sedimentation equilibrium data using non-linear least-squares methods. We discuss the implications of these results with respect to current virus assembly mechanisms.

Adenoviral E2 IVa2 protein interacts with L4 33K protein and E2 DNA-binding protein

Adenovirus (AdV) is thought to follow a sequential assembly pathway similar to that observed in dsDNA bacteriophages and herpesviruses. First, empty capsids are assembled, and then the genome is packaged through a ring-like structure, referred to as a portal, located at a unique vertex. In human AdV serotype 5 (HAdV5), the IVa2 protein initiates specific recognition of viral genome by associating with the viral packaging domain located between nucleotides 220 and 400 of the genome. IVa2 is located at a unique vertex on mature capsids and plays an essential role during genome packaging, most likely by acting as a DNA packaging ATPase. In this study, we demonstrated interactions among IVa2, 33K and DNA-binding protein (DBP) in virus-infected cells by in vivo cross-linking of HAdV5-infected cells followed by Western blot, and co-immunoprecipitation of IVa2, 33K and DBP from nuclear extracts of HAdV5-infected cells. Confocal microscopy demonstrated co-localization of IVa2, 33K and DBP in virus-infected cells and also in cells transfected with IVa2, 33K and DBP genes. Immunogold electron microscopy of purified HAdV5 showed the presence of IVa2, 33K or DBP at a single site on the virus particles. Our results provide indirect evidence that IVa2, 33K and DBP may form a complex at a unique vertex on viral capsids and cooperate in genome packaging.

Identification and characterization of a DNA binding domain on the adenovirus IVa2 protein

The adenovirus IVa2 protein has been implicated as a transcriptional activator of the viral major late promoter (MLP) and a key component in the packaging of the viral genome. IVa2 functions in packaging through its ability to form a complex with the viral L1 52/55kDa protein, which is required for encapsidation. IVa2, alone and in conjunction with another viral protein, the L4 22K protein, binds to the packaging sequence on the viral genome and to specific elements in the promoter. To define the DNA binding domain on IVa2 and determine its contribution to the viral life cycle, we created a mutant protein that lacks a putative helix-turn-helix motif at the extreme C-terminus. Characterization of this mutant protein showed that while MLP activity is relatively unaffected, it is unable to bind to and package DNA.

Cooperative Heteroassembly of the Adenoviral L4-22K and IVa2 Proteins onto the Viral Packaging Sequence DNA

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

Molecular Switching in Human Adenovirus

Human Adenovirus (Ad) is an icosahedral, double stranded DNA virus. Activating the assembly of viral particles requires at least three viral components: the IVa2 and L4-22K proteins, as well as a section of DNA within the viral genome, called the packaging sequence (PS). Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the PS, and that these interactions are absolutely required to turn on the virus assembly pathway. The IVa2 and L4-22K proteins, along with an alternatively spliced product of the L4-22K gene, called L4-33K, also function as transcriptional activators of the major late promoter (MLP).

Assembly of the Adenoviral IVa2 and L4-22K Proteins on Viral DNA Packaging Sequences

Assembly of the Adenoviral IVa2 and L4-22K Proteins on Viral DNA Packaging Sequences

Adenovirus Structural Protein IIIa Is Involved in the Serotype Specificity of Viral DNA Packaging

The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.

Characterization of Empty Adenovirus Particles Assembled in the Absence of a Functional Adenovirus IVa2 Protein

The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses-the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo. By utilizing a virus unable to express IVa2, pm8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results show that ATPase activity was not required for the assembly of empty virus particles. In addition, we present evidence that particles were assembled in the absence of IVa2 by using two viruses null for IVa2-a deletion mutant virus, ΔIVa2, and the previously described mutant virus, pm8002. Empty virus particles produced by these IVa2 mutant viruses did not contain detectable viral DNA. We conclude that the major role of IVa2 is in viral DNA packaging. A characterization of the empty particles obtained from the IVa2 mutant viruses compared to wild-type empty particles is presented.

Assembly of the Adenoviral IVa2 and L4-22K Proteins on a Viral DNA Packaging Sequence

The human adenoviral (Ad) life cycle is divided into early and late phases.In the early phase, the virus optimizes the host environment for its replication. Once the virus starts replicating its genome, the life cycle enters the late phase. Many viral proteins, including capsid proteins, are encoded in the viral late gene region and expressed during the late phase. At the end of the late phase, the viral genomes are encapsidated to form the infectious viral particles. This encapsidation process is a critical step which requires threshold levels of progeny viral genomes and capsid proteins. To optimize assembly of viral particles, the viral late gene transcription and genome encapsidation processes must proceed as ordered events, i.e. they must be precisely regulated. In this scenario, the activation of the encapsidation pathway behaves like a classic genetic switch. Previous studies have shown that two viral proteins, IVa2 and L4-22K, recognize a specific DNA binding site within both the promoter region of viral late genes and the packaging sequence (PS) DNA which are required for encapsidation. However, the DNA architecture is different in these two regions. Multiple copies of this specific binding site have been identified in the PS DNA while only a single copy has been identified in the late gene promoter. We hypothesize the interaction of IVa2, L4-22K, and these specific DNA binding sites regulates the viral decision to initiate encapsidation. To develop a predictive model to describe this process, we have initiated a physical-chemical study of the assembly of the IVa2 and L4-22K proteins on these specific DNA sequences.

Self-Association of the Adenoviral L4-22K Protein

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus that causes infections of the respiratory tract, urinary tract, and gastrointestinal tract. Assembly of virus particles requires condensation and encapsidation of the linear viral genome. This process requires sequence specific binding of two viral proteins, called IVa2 and L4-22K, to a conserved sequence located at the left end of the viral genome, called the packaging sequence (PS). IVa2 and an alternatively spliced form of L4-22K, called L4-33K, also function as transcriptional activators of the major late promoter (MLP), which encodes viral structural and core proteins. IVa2 and L4-33K bind to identical conserved DNA sequences downstream of the MLP, called the downstream element (DE), to activate transcription. To begin to dissect how the IVa2, L4-22K, and L4-33K proteins simultaneously function as transcriptional activators and DNA packaging proteins, we need to understand the thermodynamics of assembly of these proteins on DNA that contains the PS as well as the DE. Toward this end, we have characterized the self-assembly properties of highly purified, recombinant L4-22K protein. We show that L4-22K reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme. We show that the smallest polymerizing unit is likely the L4-22K monomer (s(20,w) = 2.16 ± 0.04 S) and that the monomer assembles with itself and/or other aggregates with an equilibrium association constant, L, of 112 (102, 124) μM(-1) (0.1 M NaCl, pH 7, 25 °C). A mechanistic consequence of an isodesmic, indefinite assembly process is that the free concentration of the smallest polymerizing unit cannot exceed 1/L. We discuss the implications of this observation with respect to the thermodynamics of assembly of L4-22K and IVa2 on the PS.

Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site

The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Two proteins from the adenoviral L4 unit have been suggested as DEF-A candidates. Here we have examined L4-22K and L4-33K for possible DEF-A activity. We show that L4-22K stimulates transcription from the MLP in a DE sequence dependent manner both in vivo and in vitro, and that L4-22K binds to the DE sequence in vitro. Further, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.

Adenovirus Late-Phase Infection Is Controlled by a Novel L4 Promoter

During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.