Viruses and the Nucleolus

Matthews, David A.; Emmott, Edward; Hiscox, Julian A.

doi:10.1007/978-1-4614-0514-6_14

Cited by 17 publications

(18 citation statements)

References 173 publications

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“…Taken together, although there is no inconsistency in the NLS roles of AAP2BR1 and AAP2BR2 between the AAP2 mutants and the GFP fusion proteins, their NoLS roles are context dependent. (28)(29)(30). Having created a panel of AAP2 mutants whose intracellular localizations are characterized, we addressed how aberrant localization of AAP2 might affect its assembly-promoting function.…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

Earley

Kawano

Adachi

et al. 2015

J Virol

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Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2 144 -184 ). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and ␤-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2 144 -184 has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCEAdeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolarlocalizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.A deno-associated virus (AAV) is a small, single-stranded DNA virus from the parvovirus family that has become a successful vector for gene delivery. The recent achievements in the field of AAV vector research have called attention to the incompletely understood life cycle of the v...

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Section: Resultsmentioning

confidence: 99%

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

Earley

Kawano

Adachi

et al. 2015

J Virol

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“…Over 100 viral proteins encoded by greater than 50 distinct viruses have been reported to localize to nucleoli, prompting the nucleolus to be coined the “gateway to viral infection” (Hiscox, 2002; Matthews et al, 2011). Some viruses disrupt nucleolar architecture [e.g., herpes simplex virus type 1; (Bertrand and Pearson, 2008; Lopez et al, 2008; Lymberopoulos and Pearson, 2007; Xing et al, 2010)], recruit nucleolar proteins to virus replication centers [e.g., adenovirus (Hindley et al, 2007; Lawrence et al, 2006; Okuwaki et al, 2001)], or re-localize nucleolar proteins to impair cellular metabolism [e.g., poliovirus (Waggoner and Sarnow, 1998)].…”

Section: Introductionmentioning

confidence: 99%

“…Many viral proteins that localize to nucleoli are capsid and nucleocapsid proteins that bind to viral genomes and direct virus particle assembly (Matthews et al, 2011). Most of these proteins bind RNA, so nucleolar localization has been assumed to be a consequence of the high concentration of RNA in nucleoli.…”

Section: Introductionmentioning

confidence: 99%

NC-mediated nucleolar localization of retroviral gag proteins

et al. 2013

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The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5′ untranslated RU5 region of the viral RNA genome, suggesting the psi (ψ packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that the there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection.

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“…1) that is active only in P3, indicative of specific intranuclear functions (7,(11)(12)(13). Notably, a number of viruses with cytoplasmic replication cycles express proteins able to enter the nucleus and to interact with nucleoli and core nucleolar proteins, such as nucleolin (NCL) and fibrillarin (FBL), suggestive of key roles in infection potentially related to functions in ribosome biogenesis, RNA processing, and regulation of cell cycle progression and apoptosis (14)(15)(16)(17)(18). Possible roles for nucleoli/nucleolar proteins in lyssavirus infection, however, have not been investigated.…”

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confidence: 99%

Identification of a Role for Nucleolin in Rabies Virus Infection

Oksayan

Nikolić

David

et al. 2015

J Virol

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c Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localize to nucleoli and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin and that nucleolin is important to lyssavirus infection.

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Viruses and the Nucleolus

Cited by 17 publications

References 173 publications

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

NC-mediated nucleolar localization of retroviral gag proteins

Identification of a Role for Nucleolin in Rabies Virus Infection

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