p53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G₁. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.     

RNA-Binding Activity of the E1B 55-Kilodalton Protein from Human Adenovirus Type 5

The human adenovirus 5 E1B 55-kDa protein is required for efficient nucleocytoplasmic transport of late viral mRNAs. This protein is shown to have RNA-binding activity which maps to a region of the protein with homology to a family of RNA-binding proteins and which has been shown previously to be essential for functionality of the protein in vivo.     

TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification.     

Interferon-Induced Antiviral Mx1 GTPase Is Associated with Components of the SUMO-1 System and Promyelocytic Leukemia Protein Nuclear Bodies

Mx proteins are interferon-induced large GTPases, some of which have antiviral activity against a variety of viruses. The murine Mx1 protein accumulates in the nucleus of interferon-treated cells and is active against members of the Orthomyxoviridae family, such as the influenza viruses and Thogoto virus. The mechanism by which Mx1 exerts its antiviral action is still unclear, but an involvement of undefined nuclear factors has been postulated. Using the yeast two-hybrid system, we identified cellular proteins that interact with Mx1 protein. The Mx1 interactors were mainly nuclear proteins. They included Sp100, Daxx, and Bloom's syndrome protein (BLM), all of which are known to localize to specific subnuclear domains called promyelocytic leukemia protein nuclear bodies (PML NBs). In addition, components of the SUMO-1 protein modification system were identified as Mx1-interacting proteins, namely the small ubiquitin-like modifier SUMO-1 and SAE2, which represents subunit 2 of the SUMO-1 activating enzyme. Analysis of the subcellular localization of Mx1 and some of these interacting proteins by confocal microscopy revealed a close spatial association of Mx1 with PML NBs. This suggests a role of PML NBs and SUMO-1 in the antiviral action of Mx1 and may allow us to discover novel functions of this large GTPase.     

E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p

The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.