Abstract
The L2 region of bovine adenovirus-3 (BAdV-3) encodes a Mastadenovirus genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81–120, 190–210, and 380–389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21–50 and 380–389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380–389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.
Highlights
Bovine adenovirus-3 (BAdV-3), a member of the Mastadenovirus genus, is a non-enveloped icosahedral particle, which contains a double-stranded DNA genome of 34,446 bp organized into early, intermediate, and late regions (Reddy et al, 1998)
We reported that 100K protein encoded by human adenovirus-5 (HAdV-5) and BAdV-3 differ in sub cellular localization and protein function (Makadiya et al, 2015)
Protein V is a Mastadenovirus genus specific minor core protein, which localizes to both the nucleus and the nucleolus in HAdV-5 infected cells (Matthews, 2001)
Summary
Bovine adenovirus-3 (BAdV-3), a member of the Mastadenovirus genus, is a non-enveloped icosahedral particle, which contains a double-stranded DNA genome of 34,446 bp organized into early, intermediate, and late regions (Reddy et al, 1998). The L2 region of HAdV-5 encodes a minor capsid protein named protein V (pV), which associates with the viral genome and bridges the core and the capsid proteins Expression of pV does not alter the localization of nucleolar proteins (B23, nucleolin) in infected cells, the over expression of pV redistributes nucleolin and nucleophosmin to the cytoplasm in transfected cells (Matthews, 2001). While sumoylation of pV alters the adenovirus replication, it does not change the nucleolar localization of pV in infected cells (Freudenberger et al, 2018). The nuclear localization of a protein is a well-characterized process regulated by nuclear pore complexes (NPCs) and requires active transport mechanisms, including nuclear transport receptor proteins and specific nuclear localization signal (NLS) sequences, on the transported viral protein. Nucleolar localization depends on the interactions of nucleolar constituents (proteins or rRNA) with specific viral protein sequences usually rich in arginine and lysine, which can act as the nucleolar localization signals (NoLS; Reed et al, 2006)
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