Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C pro and the viral polymerase 3D pol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3D pol at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D pol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D pol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.Poliovirus (PV), a member of the Picornaviridae family of RNA viruses, must simultaneously perform many tasks inside a host cell for efficient and successful viral replication, and only a small number of gene products are responsible for these processes. The virus produces a single polyprotein that is cleaved by virally encoded proteases. Many of the viral proteins, including precursor proteins, play multiple roles in viral replication.The viral protein 3CD is a multifunctional precursor to the poliovirus protease 3Cpro and the RNA-dependent RNA polymerase 3Dpol . The 3CD molecule retains its ability to function as a protease but lacks polymerase activity (20). Its proteolytic functions include cleavages, resulting in the production of structural proteins VP0, VP1, and VP3 and nonstructural proteins 3AB, 3CD, 3C pro , and 3D pol . In many of these functions, 3CD serves as a better protease than 3C pro , suggesting that the 3D region of 3CD contributes to that activity (34).3CD is also a crucial component of the viral replication complex. The 5Ј-terminal region of the poliovirus genome adopts a cloverleaf structure to which 3CD can bind in the presence of the viral protein 3AB or the cellular protein PCBP2 (2, 35, 49). Both forms of this ribonucleoprotein complex appear to play important roles in inducing RNA synthesis (49). The initiation of RNA synthesis is primed by the addition of two uridines to the 22-amino-acid protein primer VPg (3B) at the side-chain hydroxyl of Tyr3. Positive-strand synthesis is thought to use an RNA hairpin structure as its template within the PV genome (37,42). One model suggests that this cisacting replication signal in the 2C-noncoding region of the PV genome, cre(2C), binds to 3CD and forms a mu...

Nevoid melanoma may resemble benign compound or intradermal nevi by their silhouette and profile on low power. Higher power usually reveals nuclear atypia, confluence of cells, incomplete maturation and dermal mitotic activity. However, to some extent all of these features maybe seen in benign compound or intradermal nevi and no single criteria can be used to differentiate nevoid melanoma from a benign nevus. The distinction can be particularly problematic in nevi that show mitotic activity and we have noted a recent trend in diagnosis of melanocytic neoplasms with dermal mitosis as nevoid melanoma despite the presence of normal maturation in the dermis and lack of significant nuclear atypia. Therefore in this study we evaluated 10 nevoid melanomas, 4 of which resulted in metastasis and 10 mitotically active nevi with fluorescence in situ hybridization targeting key chromosomal loci previously shown to effectively discriminate been malignant and benign melanocytic neoplasms. All 10 nevoid melanomas showed copy number abnormalities by fluorescence in situ hybridization in either chromosome 6 or 11 while none of the 10 mitotically active nevi did. The results demonstrate that fluorescence in situ hybridization targeting key chromosomal loci on chromosomes 6 and 11 can be effective in discriminating nevoid melanomas from mitotically active nevi. Additionally, our study presents further evidence that dermal mitoses alone without other diagnostic features such as nuclear atypia and lack of maturation does not constitute sufficient evidence alone for a diagnosis of melanoma.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continues to evolve. We previously showed the HCMV-encoded G protein-coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to WT. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Furthermore, AP-1 is critical for derepression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1–independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.

The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection, and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

Human cytomegalovirus (HCMV) establishes life-long latent infection in hematopoietic progenitor cells and circulating monocytes in infected individuals. Myeloid differentiation coupled with immune dysregulation leads to viral reactivation, which can cause severe disease and mortality. Reactivation of latent virus requires chromatin reorganization and the removal of transcriptional repressors in exchange for transcriptional activators. While some factors involved in these processes are identified, a complete characterization of the viral and cellular factors involved in their upstream regulation remains elusive. Herein we show the HCMV-encoded G protein-coupled receptor (GPCR), UL33, is expressed during latency. While this viral GPCR is not required to maintain latent infection, our data reveal UL33-mediated signaling is important for efficient viral reactivation. Additionally, UL33 signaling induces cellular cAMP response element binding protein (CREB) phosphorylation, a transcription factor whose recruitment to the major immediate early (MIE) enhancer/promoter promotes reactivation. Finally, targeted pharmacological inhibition of CREB activity reverses the reactivation phenotype of the UL33 signaling deficient mutant. In sum, our data reveal UL33-mediated signaling functions to activate CREB, resulting in successful viral reactivation.