Host diversification is concurrent with linear motif evolution in a Mastadenovirus hub protein.

Over one hundred Mastadenovirus types infect seven orders of mammals. Virus-host coevolution may involve cospeciation, duplication, host switch and partial extinction events. We reconstruct Mastadenovirus diversification, finding that while cospeciation is dominant, the other three events are also common in Mastadenovirus evolution. Linear motifs are fast-evolving protein functional elements and key mediators of virus-host interactions, thus likely to partake in adaptive viral evolution. We study the evolution of eleven linear motifs in the Mastadenovirus E1A protein, a hub of virus-host protein-protein interactions, in the context of host diversification. The reconstruction of linear motif gain and loss events shows fast linear motif turnover, corresponding a virus-host protein-protein interaction turnover orders of magnitude faster than in model host proteomes. Evolution of E1A linear motifs is coupled, indicating functional coordination at the protein scale, yet presents motif-specific patterns suggestive of convergent evolution. We report a pervasive association between Mastadenovirus host diversification events and the evolution of E1A linear motifs. Eight of 17 host switches associate with the gain of one linear motif and the loss of four different linear motifs, while five of nine partial extinctions associate with the loss of one linear motif. The specific changes in E1A linear motifs during a host switch or a partial extinction suggest that changes in the host molecular environment lead to modulation of the interactions with the retinoblastoma protein and host transcriptional regulators. Altogether, changes in the linear motif repertoire of a viral hub protein are associated with adaptive evolution events during Mastadenovirus evolution.

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.

Conserved regions of bovine adenovirus-3 pVIII contain functional domains involved in nuclear localization and packaging in mature infectious virions.

Adenoviruses are non-enveloped DNA viruses that replicate in the nucleus of infected cells. One of the core proteins, named pVIII, is a minor capsid protein connecting the core with the inner surface of the capsid. Here, we report the characterization of minor capsid protein pVIII encoded by the L6 region of bovine adenovirus (BAdV)-3. Anti-pVIII serum detected a 24 kDa protein at 12-48 h post-infection and an additional 8 kDa protein at 24-48 h post-infection. While the 24 kDa protein was detected in empty capsids, only the C-terminal-cleaved 8 kDa protein was detected in the mature virion, suggesting that amino acids147-216 of the conserved C-terminus of BAdV-3 pVIII are incorporated in mature virions. Detection of hexon protein associated with both precursor (24 kDa) and cleaved (8 kDa) forms of pVIII suggest that the C-terminus of pVIII interacts with the hexon. The pVIII protein predominantly localizes to the nucleus of BAdV-3-infected cells utilizing the classical importin α/ß dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 52-72 of the conserved N-terminus bind to importin α-3 with high affinity and are required for the nuclear localization.

The coiled-coil domain of the adenovirus type 5 protein IX is dispensable for capsid incorporation and thermostability.

The 14.4-kDa hexon-associated protein IX (pIX) acts as a cement in the capsids of primate adenoviruses and confers a thermostable phenotype. Here we show that deletion of amino acids 100 to 114 of adenovirus type 5 pIX, which eliminates the conserved coiled-coil domain, impairs its capacity to self-associate. However, pIXDelta100-114 is efficiently incorporated into the viral capsid, and the resulting virions are thermostable. Deletion of the central alanine-rich domain, as in pIXDelta60-72, does not impair self-association, incorporation into the capsid, or the thermostable phenotype. These data demonstrate, first, that the self-association of pIX is dispensable for its incorporation into the capsid and generation of the thermostability phenotype and, second, that the increased thermostability results from pIX monomers binding to different hexon capsomers rather than capsid stabilization by pIX multimers.

Characterisation of the fiber gene and partial sequence of the early region 4 of bovine adenovirus 2 (short communication).

The full sequence of the fiber gene and partial sequence of the putative 17 kD protein gene of bovine adenovirus-2 (BAdV-2) were determined. The size of the fiber gene of BAdV-2 proved to be 561 amino acids, of which the amino acids 37 to 385 form a typical shaft domain of 22 repetitive motifs. On the complementary strand, a gene homologous to the 17 kD protein coded in the E4 region of several human adenoviruses was found. The sequence analysis seems to confirm the presence of an intron in the sequenced part of the E4 region.

Comparison of the deduced amino acid sequence of guinea pig adenovirus hexon protein with that of other mastadenoviruses.

PURPOSE: We sought to isolate, clone, and determine the nucleic acid sequence of the guinea pig adenovirus (GPAdV) hexon gene. From this, the amino acid sequence of the cloned portion was deduced and compared with a set of mastadenovirus hexons. METHODS: The DNA isolated from a histologic section of infected guinea pig lung was subjected to high-fidelity amplification, using degenerate primers complementary to a conserved nucleic acid sequence near the 3' end of the hexon gene of mastadenoviruses and a 5' primer from GenBank accession No. X95630 (GPAdV hexon gene partial sequence). The amplified product was cloned, the nucleic acid sequence was determined, and the amino acid sequence was deduced and compared with the hexon amino acid sequences of 25 mastadenoviruses. RESULTS: The cloned fragment comprised 1,603 base pairs (bp) [approximately 50%]) of the hexon. Of the initial 278 nucleic acids of the clone, 276 were identical with GenBank accession No. X95630, and the deduced amino acid sequences of both were identical. The deduced GPAdV hexon amino acid sequence from the clone aligned with structural regions NT, V1, DE1, and FG1 described for human adenovirus types 2 and 5. The GPAdV hexon had < 50% similarity in amino acid sequence, compared with hexons of 25 other mastadenoviruses. Analysis of regional peptide similarities revealed the GPAdV hexon to be more similar to animal mastadenoviruses and human subgroups A, C and F than to other human subgroups. CONCLUSIONS: The cloned portion of the GPAdV hexon contained a sequence nearly identical to that of GenBank accession No. X95630. Compared with the truncated amino acid sequences of human adenovirus types 2 and 5, the deduced GPAdV hexon amino acid sequence was similar in areas structurally conserved, but different in areas associated with type-specific antigenicity.

[The patterns in the molecular biology of the adenoviruses (the results of a 20-year study at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine)]. / Nekotorye zakonomernosti molekuliarnoi biologii adenovirusov (itogi 20-letnego izlucheniia v Institute mikrobiologii i virusologii NAN Ukrainy).

Scientific data of the highest importance and priority concerning regularities of structural and functional organization of proteins of adenoviruses capsids and peculiarities of expression of the virus genome are as follows: New antigen determinants of hexon and adenovirus fiber have been discovered, their different nature (conformational or linear) and different orientation, depending on the spatial organization of proteins, have been proved; localization of some epitopes has been determined with the help of synthesised antigen-active peptides, imitating them. Some regularities of structural and functional organization of adenovirus hexon have been determined on the basis of comparative analysis of antigenic specificity and primary structure of proteins being apart in taxonomic respect. The conception of immunoactivation (infectivity neutralization) of adenoviruses has been developed, and a mathematical model of this process has been first proposed, which determines the impact of antibodies to several antigenic determinants of hexon and fiber as well as interferon and complement. The unknown peculiarities of the adenovirus genome expression were studied in the dynamics of productive infection or under the effect of modified nucleosides, proteolysis inhibitors and those of different nature promising for chemotherapy of adenovirus infection. Lymphotropicity of adenoviruses was established and a model of the mixed infection of lymphocytes with adenoviruses, HIV, and Epstein-Barr virus of the herpes virus family was proposed for the first time. It was determined that the mutual interference of viruses was developed at the process of a single or successive infection and this was important to understand AIDS immunopathogenesis. Data presented substantiate the ways of creation of modern efficient preparations for diagnosis, prophylaxis and chemotherapy of adenovirus infection.

Completion of the DNA sequence of mouse adenovirus type 1: sequence of E2B, L1, and L2 (18-51 map units).

The DNA sequence of 9991 nt, corresponding to 18-51 map units of mouse adenovirus type 1 (MAV-1), was determined, completing the sequence of the Larsen strain of MAV-1. The length of the complete MAV-1 genome is 30,946 nucleotides, consistent with previous experimental estimates. The 18-51 map unit region encodes early region 2B proteins necessary for adenoviral replication as well as late region L1 and L2 structural and packaging proteins. Sequence comparison in this region with human adenoviruses indicates broad similarities, including colinear preservation of all recognized open reading frames (ORFs), with highest amino acid identity occurring in the DNA polymerase and polypeptide III (penton base subunit) ORFs. Virus-associated (VA) RNA is not encoded in the region where VA RNAs are found in the human adenoviruses, between E2B and L1, nor is it encoded anywhere in the entire MAV-1 genome. The MAV-1 polypeptide III lacks the arginine-glycine-aspartic acid (RGD) motif which is involved in an association with cell-surface integrins. Only one RGD sequence is found in an identified coding region in the entire MAV-1 genome. Similar to the porcine adenovirus, this RGD sequence is found in the C-terminus of the MAV-1 fiber protein.

Hydropathic characteristics of adenovirus hexons.

The complete nucleotide sequence and the predicted amino acid sequence of the adenovirus type 7 hexon gene were determined. The hydropathy of the hexon proteins from human adenovirus types 2, 3, 4, 5, 7, 12, 16, 40, 41, and 48, bovine adenovirus type 3, murine adenovirus type 1, and avian adenovirus types 1 and 10 was analysed. The presence of purines and pyrimidines in the second position of the codons was correlated to hydrophilicity and hydrophobicity, respectively. Comparison of the hydrophilicity plots of eight hexons showed seven hypervariable regions to be distributed on the surface. A large portion of the hypervariable regions manifests hydrophilicity. The strength of the surface charge accumulated on the hydrophilic and hydrophobic regions correlated to the tissue tropism of the different adenovirus types. Analysis of codon usage for adenovirus hexons showed that among synonymous codons those with cytidine in the third position were preferably used to a great extent. Analysis of the nucleotide and amino acid sequence pair distances and the phylogenetic tree of 14 hexon proteins showed members of subgenera B, D and E to be closely related, especially Ad4 and Ad16, and subgenus A to be closely related to subgenus F.

Sequence analysis of the fiber genomic region of a porcine adenovirus predicts a novel fiber protein.

The complete nucleotide sequence of the putative fiber protein of a porcine adenovirus isolate, NADC-1, was determined. The coding sequence for the fiber protein was found to be 2112 nucleotides, predicting a 703 amino acid protein with a calculated molecular mass of 76,681 Da. The coding sequence is located between 86 and 92.5 map units. A polyadenylation signal was found 44 bases downstream of the stop codon. Northern hybridization analysis identified a band at 2.4 kb, which likely includes the primary transcript plus a tripartite leader (typically found with adenovirus transcripts) and a polyA(+) tail. The predicted NADC-1 fiber protein was found to have a tail domain comparable in size and sequence to most adenovirus fiber proteins, a comparatively short shaft region, and a head region that was more than twice as large as that of other adenovirus fiber proteins. Each of the three structural domains (tail, shaft, and head) of the predicted NADC-1 fiber protein was found to be most similar to the corresponding domain of a different mammalian adenovirus. The NADC-1 fiber head contained an RGD sequence, a motif that is found in the penton protein of other adenoviruses. Furthermore, the predicted amino acid sequence of the C-terminal half of the NADC-1 fiber head has significant homology to S-lectin proteins, a characteristic not shared with other adenovirus fiber proteins. Thus the predicted amino acid sequence of the NADC-1 fiber head is unique among adenoviruses for which the sequence of the fiber protein is known.

Adenovirus protein-protein interactions: hexon and protein VI.

A variety of mastadenoviruses were denatured, their polypeptides separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose. The immobilized polypeptides were washed, incubated with buffers containing hexons from human adenoviruses (Ad) types 2, 5 and 12 and the location of bound hexons was detected with anti-hexon antibodies. It was found that hexons from any of the three human adenovirus types bound to protein VI from all the mastadenoviruses examined. Furthermore we found that hexon-VI binding was significantly greater than the interaction between hexon and the precursor to VI, pVI. This binding was susceptible to detergents and to changes in pH or salt concentration. A rabbit polyclonal antibody was raised against a recombinant protein derived from the middle third of pVI from Ad2 and was used to quantify the difference in binding and to demonstrate the presence of a single intermediate (designated iVI) in the processing of pVI to VI. The affinity between iVI and hexon was considerably greater in our assay than that of pVI but was less than that between hexon and VI. A complementary binding of recombinant iVI to immobilized hexons was also demonstrated. This latter interaction, however, was only observed when hexon preparations were not boiled prior to electrophoresis, substantiating the proposition that the recognition motif on the hexon was conformation-dependent. These results are discussed in the context of understanding further the molecular basis of protein-protein interactions between the structural proteins of adenoviruses and the factors involved in virion maturation.

The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 A resolution.

The crystal structure of hexon, the major coat protein from adenovirus type 2, has been refined at 2.9 A resolution. Hexon is a homo-trimer (molecular mass 3 x 109,077 Da) and crystallizes in the cubic space group P2(1)3, with a cell edge of 150.5 A. There are four molecules in the unit cell so that the crystallographic asymmetric unit contains one subunit of the trimer. The electron density in most regions is well-defined and 880 amino acid residues, of the 967 in this unusually long polypeptide chain, have been located and fitted. The N terminus (1 to 43) and three internal stretches (192 to 203, 270 to 291 and 444 to 453) are not defined, and a stretch (168 to 207) with unclear side-chain density is modelled as poly(Ala/Gly). The current refined model, consisting of 6943 non-hydrogen protein atoms and 85 water molecules, yields an R-factor of 19.9% for 18,176 reflections in the resolution range 5.0 to 2.9 A. The model has reasonable geometry with root-mean-square deviations from ideal bond lengths of 0.022 A and angle-related 1-3 distances of 0.056 A. The overall shape of the trimeric hexon molecule is unusual and may be divided into a pseudo-hexagonal base rich in beta-structure, and a triangular top formed from three long loops containing some secondary structure. The base contains two similar pedestal domains, P1 and P2, each of which is a flattened eight-stranded beta-barrel with the "jelly-roll greek key" topology characteristic of other viral coat proteins. P1 and P2 are related by an approximate 6-fold operation about the molecular 3-fold axis so that six barrels form the walls of the tubular hexon base. The hexon bases form close-packed p3 arrays on each facet of the icosahedral adenovirus virion. Unlike other viral capsids, the barrel axes are almost perpendicular to rather than parallel with the capsid surface. The hexon top, which consists of intimately interacting loops emerging from P1 and P2 in the base, has a triangular outline and so does not exhibit the pseudo-symmetry of the base. The structure of the hexon trimer shows how economically it meets the demands of its function as a stable protective viral coat, reveals the significance of the special features in its unusual amino acid sequence, and explains its biochemical and immunological properties. The molecule is hollow, with a large central cavity, and so has a high effective volume for its mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Sequence analysis of the putative E3 region of bovine adenovirus type 2.

Adenoviruses are nonenveloped icosahedral-shaped particles. The double-stranded viral DNA genome contains four major early transcription units, designated E1 (a and b), E2 (a and b), E3 and E4, which are expressed in a regulated manner soon after infection. The gene products of the region E3, shown to be nonessential for viral replication in vitro, are believed to be involved in counteracting host immunosurveillance. Human adenovirus type 5 DNA sequences of transcription units L4 and L5 adjacent to E3 were used to localize E3 within the bovine adenovirus type 2. The DNA sequences between 74.8 and 84.4 mu containing E3 and the fiber gene were determined. The E3 region was found to consist of about 2.3 kb pairs and to encode four proteins longer than 60 amino acids. However, these four open reading frames did not show significant homology to any other known adenovirus DNA or protein sequence.

Sequence analysis of bovine adenovirus type 2 early region 1 and pIX gene.

Bovine adenoviruses (BAVs) cause both respiratory and/or enteral diseases in cattle and are widespread throughout the world. We have sequenced the extreme left end (0 to 12.2 mu) of the BAV2 genome. Partial analysis of the nucleotide sequence revealed 12 potential open reading frames (ORFs) which could encode for polypeptides of 50 or more amino acids. These ORFs were compared to those of the early region 1 (E1) proteins and hexon-associated protein IX (pIX) from other adenoviruses (Ads). The 4 major ORFs show homology to known Ad polypeptides. The 4 BAV2 ORFs are located in approximately the same area as that of the human Ad type 5 (HAd5) E1A, E1B and pIX ORFs. ORF1 has the potential to encode a 119-amino-acid-long polypeptide that is 85.7% homologous (over 42 amino acids) to the E1A conserved region III of both HAd2 and HAd5. ORF2 and ORF3 have the potential to code for 160- and 408-amino-acid-long polypeptides, respectively. The 160-amino-acid-long polypeptide exhibits 71.3% homology to the small T antigen of HAd40, and the 408-amino-acid polypeptide exhibits 67.0% homology with the large T antigen of HAd2. Both the small T antigen of HAd40 and the large T antigen of HAd2 are translated from the E1B mRNAs. ORF4 has the potential to code for a 117-amino-acid-long polypeptide that has 77.9% homology with the Tupaia adenovirus pIX.

Sequence and structural analysis of murine adenovirus type 1 hexon.

The genomic region encoding the major capsid protein (hexon) of murine adenovirus type 1 (MAV-1) has been isolated and sequenced. The sequence predicts a 908 residue MAV-1 hexon protein and is flanked by a portion of the upstream pVI gene and the downstream endoproteinase gene. The order of these genes and their location in the middle of the genome are the same as those found in other adenoviruses sequenced to date. Multiple sequence alignment with the other five known hexon protein sequences reveals an overall residue identity of 51% and residue conservation of 66%. In comparison with human adenovirus type 2 (Ad2), MAV-1 hexon has major deletions between residues 141 to 170, 270 to 284 and 446 to 455. Since these regions in the Ad2 hexon are partially exposed on the outer surface of the virion, they may represent type-specific antigenic determinants. The MAV-1 hexon sequence has been modelled using the known three-dimensional structure of the Ad2 hexon. The variable regions in which the mutations, deletions and insertions occur are located in the l1 and l2 loops of the molecule that form the protruding hexon towers on the external surface of the virion.