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/β 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.

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Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX

Virology ◽

10.1016/j.virol.2003.12.007 ◽

2004 ◽

Vol 320 (2) ◽

pp. 291-300 ◽

Cited By ~ 22

Author(s):

Alexander Zakhartchouk ◽

Wayne Connors ◽

Andrew van Kessel ◽

Suresh Kumar Tikoo

Keyword(s):

Capsid Protein ◽

Heterologous Protein ◽

Adenovirus Type ◽

Minor Capsid Protein ◽

Bovine Adenovirus ◽

C Terminus ◽

Protein Ix ◽

Bovine Adenovirus Type 3 ◽

Type 3

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Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus

Journal of General Virology ◽

10.1099/0022-1317-83-12-3093 ◽

2002 ◽

Vol 83 (12) ◽

pp. 3093-3102 ◽

Cited By ~ 86

Author(s):

Shao-Hung Wang ◽

Wan-Jr Syu ◽

Kao-Jean Huang ◽

Huan-Yao Lei ◽

Chen-Wen Yao ◽

...

Keyword(s):

Dengue Virus ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Core Protein ◽

Intracellular Localization ◽

Intracellular Location ◽

The Core ◽

Core Proteins ◽

Localization Signal ◽

Infected Cells

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6–9, and mutation of the second one (KKSK), located at aa 73–76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85–100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

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The coordinating role of the human norovirus minor capsid protein VP2 is essential to functional change and nuclear localization of the major capsid protein VP1

Archives of Virology ◽

10.1007/s00705-019-04192-2 ◽

2019 ◽

Vol 164 (4) ◽

pp. 1173-1180 ◽

Cited By ~ 2

Author(s):

Zhili Liu ◽

Min Zhang ◽

Zhen Shen ◽

Huifen Chen ◽

Wanju Zhang ◽

...

Keyword(s):

Capsid Protein ◽

Nuclear Localization ◽

Major Capsid Protein ◽

Functional Change ◽

Human Norovirus ◽

Minor Capsid Protein ◽

Capsid Protein Vp1

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Interaction of the Influenza A Virus Polymerase PB2 C-terminal Region with Importin α Isoforms Provides Insights into Host Adaptation and Polymerase Assembly

Journal of Biological Chemistry ◽

10.1074/jbc.m110.182964 ◽

2011 ◽

Vol 286 (12) ◽

pp. 10439-10448 ◽

Cited By ~ 50

Author(s):

Stephane Boivin ◽

Darren J. Hart

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Influenza A ◽

Host Adaptation ◽

New Host ◽

Adaptive Mutations ◽

C Terminus ◽

Importin Α ◽

Localization Signal ◽

Import Receptors

In the adaptation of avian viruses to mammalian hosts, mutations in the viral polymerase, notably in the PB2 subunit, play an important role. A PB2 C-terminal domain rich in putative host adaptation residues has been shown to bind importin α nuclear import receptors. Adaptation has been proposed to involve binding of PB2 to importins of the new host. To date PB2-importin complexes have been characterized semiquantitatively with no precise measurement of binding parameters. To investigate the effects of adaptive mutations on importin interaction and selectivity, surface plasmon resonance was used to compare the binding rate constants and affinities of avian H5N1 and human H3N2 PB2 C-terminal variants with importin isoforms human α 1, 3, 5 and 7, and avian α 1. Using purified proteins eliminates host environment effects and permits measurement of intrinsic affinities and rates of complex formation and dissociation. Two effects were observed: first, adaptive mutations D701N, R702K, and S714R in the nuclear localization signal domain increased 2–4-fold the association rates with avian and human importins; second, measurement of different structural forms of the PB2 C terminus demonstrated that the upstream 627 domain reduced binding affinity, consistent with a steric clash predicted from crystal structures. From these kinetic data, structural analyses, and the data of others, a model is proposed in which an increase in charged surface residues during host adaptation increases the association rate of PB2 to cytoplasmic importins and where the C-terminal 627-nuclear localization signal domain may reorganize upon importin binding, consistent with a role in active polymerase assembly.

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Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1148-1155.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1148-1155

Author(s):

L D Fresco ◽

M G Kurilla ◽

J D Keene

Keyword(s):

Vesicular Stomatitis Virus ◽

Rna Binding ◽

Core Protein ◽

Protein A ◽

Core Complex ◽

The Core ◽

Core Proteins ◽

Infected Cells ◽

Vesicular Stomatitis ◽

Small Nuclear Ribonucleoproteins

After infection of baby hamster kidney cells with vesicular stomatitis virus (VSV), processing and assembly of small nuclear ribonucleoproteins (snRNP) were rapidly inhibited. The U1 and U2 snRNAs accumulated as precursor species approximately 3 and 10 nucleotides longer, respectively, than the mature RNAs. Alteration in snRNP assembly was noted because the precursor snRNAs were not associated with the U-series RNA-core protein complex in infected cells. However, antibodies specific for the U2 RNA-binding protein, A', were able to precipitate pre-U2 RNAs from VSV-infected cells. These results indicated that precursors to U2 RNA were bound to A' and remained bound during virus infection. Analysis of the synthesis of proteins normally associated with U1 and U2 RNAs indicated that synthesis was unaffected at times when snRNP assembly with core proteins was blocked by the VSV. These findings suggested that the core proteins associate with one another in the absence of the snRNAs in VSV-infected cells. They further suggest a correlation between the inability of the core complex to bind the U-series snRNPs and the failure to process the 3' ends of U1 and U2 RNAs in VSV-infected cells. These effects of VSV on snRNP assembly may be related to the shutoff of host-cell macromolecular synthesis.

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An aromatic amino acid and associated helix in the C-terminus of the potato leafroll virus minor capsid protein regulate systemic infection and symptom expression

PLoS Pathogens ◽

10.1371/journal.ppat.1007451 ◽

2018 ◽

Vol 14 (11) ◽

pp. e1007451 ◽

Cited By ~ 6

Author(s):

Yi Xu ◽

Washington Luis Da Silva ◽

Yajuan Qian ◽

Stewart M. Gray

Keyword(s):

Amino Acid ◽

Capsid Protein ◽

Aromatic Amino Acid ◽

Systemic Infection ◽

Potato Leafroll Virus ◽

Symptom Expression ◽

Minor Capsid Protein ◽

C Terminus ◽

Leafroll Virus

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The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

Journal of Virology ◽

10.1128/jvi.02057-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1178-1189 ◽

Cited By ~ 2

Author(s):

Li-Chen Yen ◽

Jia-Teh Liao ◽

Hwei-Jen Lee ◽

Wei-Yuan Chou ◽

Chun-Wei Chen ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Virus Replication ◽

Japanese Encephalitis ◽

Protective Immunity ◽

Nonstructural Protein ◽

Encephalitis Virus ◽

The Core ◽

Nonstructural Protein 1 ◽

C Terminus ◽

Infected Cells

ABSTRACTNS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using atrans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain.IMPORTANCEThe positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

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Protective Immunity to Rabbit Oral and Cutaneous Papillomaviruses by Immunization with Short Peptides of L2, the Minor Capsid Protein

Journal of Virology ◽

10.1128/jvi.76.19.9798-9805.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 9798-9805 ◽

Cited By ~ 73

Author(s):

Monica E. Embers ◽

Lynn R. Budgeon ◽

Martin Pickel ◽

Neil D. Christensen

Keyword(s):

Capsid Protein ◽

Protective Immunity ◽

Neutralizing Antibody ◽

Distinct Region ◽

Minor Capsid Protein ◽

Cottontail Rabbit ◽

Infected Cells ◽

Hpv Types

ABSTRACT The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.

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