Dehydroalanine is a synthetic precursor to a wide array of protein modifications. We describe multiple methods for the chemical conversion of cysteine to dehydroalanine on peptides and proteins. The scope and limitations of these methods were investigated with attention paid to side reactions, scale, and aqueous-and bio-compatibility. The most general method investigated-a bis-alkylation-elimination of cysteine to dehydroalanine-was applied successfully to multiple proteins and enabled the siteselective synthesis of a glycosylated antibody.Scheme 1 Dehydroalanine is a precursor to modified peptides and proteins.

Human papillomaviruses (HPVs) are small DNA viruses causally associated with benign warts and multiple cancers, including cervical and head-and-neck cancers. While the vast majority of people are exposed to HPV, most instances of infection are cleared naturally. However, the intrinsic host defense mechanisms that block the early establishment of HPV infections remain mysterious. Several antiviral cytidine deaminases of the human APOBEC3 (hA3) family have been identified as potent viral DNA mutators. While editing of HPV genomes in benign and premalignant cervical lesions has been demonstrated, it remains unclear whether hA3 proteins can directly inhibit HPV infection. Interestingly, recent studies revealed that HPV-positive cervical and head-and-neck cancers exhibited higher rates of hA3 mutation signatures than most HPV-negative cancers. Here, we report that hA3A and hA3B expression levels are highly upregulated in HPV-positive keratinocytes and cervical tissues in early stages of cancer progression, potentially through a mechanism involving the HPV E7 oncoprotein. HPV16 virions assembled in the presence of hA3A, but not in the presence of hA3B or hA3C, have significantly decreased infectivity compared to HPV virions assembled without hA3A or with a catalytically inactive mutant, hA3A/E72Q. Importantly, hA3A knockdown in human keratinocytes results in a significant increase in HPV infectivity. Collectively, our findings suggest that hA3A acts as a restriction factor against HPV infection, but the induction of this restriction mechanism by HPV may come at a cost to the host by promoting cancer mutagenesis. IMPORTANCEHuman papillomaviruses (HPVs) are highly prevalent and potent human pathogens that cause >5% of all human cancers, including cervical and head-and-neck cancers. While the majority of people become infected with HPV, only 10 to 20% of infections are established as persistent infections. This suggests the existence of intrinsic host defense mechanisms that inhibit viral persistence. Using a robust method to produce infectious HPV virions, we demonstrate that hA3A, but not hA3B or hA3C, can significantly inhibit HPV infectivity. Moreover, hA3A and hA3B were coordinately induced in HPV-positive clinical specimens during cancer progression, likely through an HPV E7 oncoprotein-dependent mechanism. Interestingly, HPV-positive cervical and head-and-neck cancer specimens were recently shown to harbor significant amounts of hA3 mutation signatures. Our findings raise the intriguing possibility that the induction of this host restriction mechanism by HPV may also trigger hA3A-and hA3B-induced cancer mutagenesis. Human papillomaviruses (HPVs) are small, nonenveloped DNA viruses known as one of the most prevalent sexually transmitted pathogens. Among almost 200 different genotypes, ϳ24 high-risk HPV genotypes are causally associated with multiple human cancers, including nearly all cervical cancers and a portion of head-and-neck squamous cell carcinomas (1). From 1988 to 2004, the incidence of HPV-associ...

Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

Human papillomavirus (HPV) infection is severely limited in its natural host, primary human keratinocytes. Our data show HPV infectivity in primary keratinocytes is over 100- and 1,000-fold lower than in established keratinocyte cell lines NIKS and HaCaT, respectively. Here, we show that the basal level of autophagy in primary human foreskin keratinocytes (HFKs) is higher than in immortalized keratinocytes, and that HPV16 virions significantly induce autophagy in HFKs. Interestingly, HPV16 infectivity is dramatically enhanced by knockdown of essential autophagy genes as well as biochemical inhibition of autophagy. The increase in HPV16 infectivity by autophagy inhibition is most significant in HFKs, showing an inverse correlation with basal HPV16 infectivity in HFK, NIKS, HaCaT, and 293FT cells. Further, inhibition of autophagy delays degradation of HPV16 capsid proteins during virus trafficking, indicating that host autophagy induced by HPV16 virions inhibits infection of primary keratinocytes through rapid degradation of viral capsid proteins.

Type I interferons (IFN-α and β) induce dynamic host defense mechanisms to inhibit viral infections. It has been recently recognized that the interferon-inducible transmembrane proteins (IFITM) 1, 2 and 3 can block entry of a broad spectrum of RNA viruses. However, no study to date has focused on the role of IFITM proteins in DNA virus restriction. Here, we demonstrate that IFN-α or -β treatment of keratinocytes substantially decreases human papillomavirus 16 (HPV16) infection while robustly inducing IFITM1, 2 and 3 expression. However, IFITM1, 2 and 3 overexpression did not inhibit HPV16 infection; rather, IFITM1 and IFITM3 modestly enhanced HPV16 infection in various cell types including primary keratinocytes. Moreover, IFITM1, 2 and 3 did not inhibit infection by two other DNA viruses, human cytomegalovirus (HCMV) and adenovirus type 5 (Ad5). Taken together, we reveal that the entry of several DNA viruses, including HPV, HCMV, and Ad5 is not affected by IFITM1, 2 and 3 expression. These results imply that HPV, and other DNA viruses, may bypass IFITM restriction during intracellular trafficking.