We have investigated the DNA-binding, oligomerization, and trans-activation functions of isolated segments of murine p53. We find that p53 has two autonomous DNA-binding regions. One domain, from amino acid 280 to 390, forms stable tetramers and binds DNA nonspecifically. The biological significance, ff any, of this DNA-binding activity is not known. A second domain, from amino acid 80 to 290, does not form stable tetramers under stringent conditions but binds DNA both specifically and nonspecifically. The specific DNA-binding function of p53, therefore, resides in the highly conserved central region of the protein and does not require stable tetramerization. Amino acids 1-290, which include both the specific DNA-binding domain and the amino-terminal acidic region, activate a p53-specific promoter in vivo. This finding strongly argues that the DNA-binding activity of p53 segment 80-290 is physiologically significant. The role of tetramerization in p53 function remains to be determined.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-β and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread.

We have analyzed the specific interaction of murine p53 with the consensus DNA-binding sequence 5-AGACATGCCT-AGACATGCCT-3. We used segments of p53 lacking the C-terminal, nonspecific DNA-binding domain because the presence of an autonomous nonspecific DNA-binding domain in wild-type p53 would complicate analysis of site-specific DNA binding. p53 amino acids 1 to 360 bind the consensus sequence as tetramers, and DNA binding promotes tetramer-tetramer interactions. p53 amino acids 80 to 290, lacking both the nonspecific DNA-binding and tetramerization domains, consistently bind consensus DNA as four monomers and only as four monomers. The virtual absence of stable binding by fewer than four monomers, even at low concentrations of p53, argues that binding by amino acids 80 to 290 is strongly cooperative. Because p53 tetramers and monomers do not simultaneously bind a single DNA consensus sequence, we conclude that a single tetramer of wild-type p53 engages the recognition sequences of the entire DNA consensus site. We further show that consensus DNA consists of two functional half-sites. Insertions, deletions, or rearrangements within the half-sites reduce DNA binding dramatically. In contrast, two half-sites separated by insertions bind p53 relatively efficiently. Insertions that place half-sites on opposite faces of the DNA helix reduce DNA binding more than insertions that place half-sites on the same face of the helix. Transcription studies, in vivo, strongly confirm the rotational specificity of the p53 interaction with consensus DNA. The ability of single p53 tetramers to bind separated DNA half-sites argues that p53 has a flexible tetramerization region.Specific DNA binding and transcriptional activation play central roles in the suppression of cellular proliferation by p53 (6,29,32). Most p53 mutations that are associated with human cancer affect the central conserved region of p53 (13) that is necessary and sufficient for specific DNA binding (2, 17, 31). Indeed, many mutations severely reduce site-specific DNA binding (1, 15) and transactivation in vivo (9,16,20). In model systems, there is also an excellent correlation between the transactivation and suppression functions of p53 (19,21). Removal of the natural transactivation domain of p53 blocks suppression of transformation by other oncogenes. Replacement of the transactivation domain with the heterologous VP16 transactivation domain restores transactivation and suppression.Studies of human and murine p53 have identified a number of autonomous functional domains. The N-terminal, acidic region strongly activates transcription when positioned at a promoter either as part of p53 or as a chimera with the GAL4 DNA-binding domain (10,20). The large central conserved region of p53 binds specific DNA sequences (2, 17, 31) and forms unstable oligomers without a preference for a particular oligomeric form (30). The C-terminal region assembles stable tetramers (30) and binds to DNA without apparent specificity (31). Surprisingly, C-terminal-truncation mutants lac...

Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. (4,7,13). In contrast, many mutant p53s enhance transformation under the same conditions. These findings argue that mutant p53 interferes with the suppression functions of endogenous WT p53. Because p53 forms oligomeric structures, the dominant negative phenotype of the mutants has been attributed to the formation of mixed WT and mutant oligomers (12). In support of this hypothesis, the C-terminal region of p53 has both oligomerization and transformation functions (18,22,23,28). The oligomerization domains of p53, however, have not been mapped in detail.Although the suppression functions of p53 are not completely understood, one mechanism by which p53 acts is the direct activation of transcription via site-specific DNA binding (5, 9, 15). Presumably, p53 activates genes that inhibit cellular proliferation such as the WAF] (Cipl) gene (3,6,11). The role of p53 oligomerization in DNA binding and transactivation is not clear. Recently, we and others showed that p53 has two autonomous DNA-binding regions (1,20,26,29 (segment 80-290), does not form stable tetramers but binds DNA specifically. Furthermore, amino acids 1 to 290, which include both the specific DNA-binding domain and the Nterminal acidic region, activate a p53-specific promoter in vivo. The specific DNA binding and transactivating functions of p53, therefore, do not require stable tetramerization. Because the DNA recognition site for p53 consists of four imperfect repeats, however, it would be surprising if oligomerization did not contribute to the DNA binding function of p53.We have undertaken the present study to define further the oligomerization properties of p53 and the role of oligomerization in cellular transformation. There has been some disagreement about the nature of the various quaternary structures formed by p53. This confusion is the result of a number of factors. First, purified p53 consists of many oligomeric forms that require high-resolution techniques for analysis. Second, p53 appears to have an unusual shape that leads to anomalous behavior during gel filtration (8). Finally, murine and human pS3s have been studied independently but have not been directly compared. We have, therefore, used a variety of techniques to define the quaternary structures of murine and human p53s. The availability of purified segments of p53 has allowed us to separate two autonomous oligomerization domains of the protein. We find that murine amino acids 315 to 350 and human amino acids 323 to 355 encode a strong tetramerization domain. Murine amino acids 80 to 320 and human amino acids 83 to 323 form unstable oligomers without a preferred species. We also present evidence that the C...