Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V

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.

Adenovirus Core Proteins: Structure and Function

“…However, a recent report suggests that although pV is bound to the incoming viral genome, it dissociates from the virus partly at the entry of the AdV core in the cytoplasm and at the nuclear pore complex [59] without entering the nucleus. In contrast, newly synthesized pV actively transports to the nucleus with the help of its own nuclear localization signals (NLSs) by utilizing α/β nuclear import receptor-mediated nuclear transport machinery [76,77]. While a number of studies have reported the presence of both monopartite [76,77] and/or bipartite [76] NLSs, the location of NLSs and use of nuclear receptor for nuclear transport of pV may differ among pV encoded by different members of mAdV [38,76,77].…”

“…In contrast, newly synthesized pV actively transports to the nucleus with the help of its own nuclear localization signals (NLSs) by utilizing α/β nuclear import receptor-mediated nuclear transport machinery [76,77]. While a number of studies have reported the presence of both monopartite [76,77] and/or bipartite [76] NLSs, the location of NLSs and use of nuclear receptor for nuclear transport of pV may differ among pV encoded by different members of mAdV [38,76,77].…”

“…Efficient replication of some viruses, including AdV, induces alterations in the nucleolus [76,78,79], which requires viral proteins to localize to the nucleolus. Interestingly, pV also localizes to the nucleolus of both infected and transfected cells [76,77] though pV does not accumulate in the nucleolus [38]. Deletion mutant analyses suggested that pV encoded by different mAdVs contain two independent nucleolar localization motifs, which appear to be functionally redundant [76,77] and may utilize transportin for nucleolar localization [38].…”

“…Interestingly, pV also localizes to the nucleolus of both infected and transfected cells [76,77] though pV does not accumulate in the nucleolus [38]. Deletion mutant analyses suggested that pV encoded by different mAdVs contain two independent nucleolar localization motifs, which appear to be functionally redundant [76,77] and may utilize transportin for nucleolar localization [38]. Interestingly, the production of stable infectious virion requires the presence of both NoLSs [76].…”

“…Interestingly, the production of stable infectious virion requires the presence of both NoLSs [76]. While nucleolar localization of HAdV-5 pV induces translocation of B23.1 and nucleolin in transiently overexpressed transfected cells [76], the nucleolar localization of BAdV-3 pV does not alter the nucleolar distribution of B23.1 or nucleolin in infected/transfected cells [77]. Remarkably, pV encoded by mAdVs appears to contain both non-overlapping and overlapping NLS/NoLS [76,77].…”