As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an “immature” nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a “maturation signal” that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, “Single Strand Blocking” model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis.

Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.T he human hepatitis B virus (HBV) continues to pose a significant health risk worldwide, causing more than one million deaths annually (52). Chronic HBV infection, estimated to affect 350 million people globally, dramatically elevates the risk for developing serious liver diseases, including cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family, which includes hepatotropic DNA viruses that consist of an enveloped icosahedral capsid enclosing an approximately 3-kb DNA genome in a partially double-stranded, relaxed circular (RC) form. These DNA viruses are also retroid viruses and encode a reverse transcriptase (RT) enzyme that converts a so-called pregenomic RNA (pgRNA) template to the RC DNA through reverse transcription within cytoplasmic capsids. Capsids are composed of multiple copies (180 or 240) of one virally encoded protein, the core or capsid protein (9,63,65,71).Phosphorylation of the hepadnavirus core protein is important for RNA packaging, DNA synthesis, and subcellular localization. The HBV core protein (HBc) contains three major serine-proline (S-P) phosphorylation sites in its C-terminal domain (CTD) (32). The duck hepatitis B virus (DHBV) core protein (DHBc) contains six known phosphorylation sites, four of which also have the serine/threonine-proline (S/T-P) motifs (43, 68). Mutational analyses indicate that phosphorylation of the core protein at these S/T-P sites is required for RNA packaging and DNA synthesis in HBV (29, 31). For DHBV, dynamic CTD phosphorylation at the S/T-P sites is required for complete DNA synthesis such that the S/T-P phosphorylation is needed for first-strand DNA synthesis and dephosphorylation is required for second-strand DNA s...

Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions.

The hepadnavirus reverse transcriptase (RT) has the unique ability to initiate viral DNA synthesis using RT itself as a protein primer. Protein priming requires complex interactions between the N-terminal TP (terminal protein) domain, where the primer (a specific Y residue) resides, and the central RT domain, which harbors the polymerase active site. While it normally utilizes the cis-linked TP to prime DNA synthesis (cis-priming), we found that the duck hepatitis B virus (DHBV) RT domain, in the context of the full-length RT protein or a mini-RT construct containing only truncated TP and RT domains, could additionally use a separate TP or RT domain in trans as a primer (trans-priming). trans interaction could also be demonstrated by the inhibitory effect (trans-inhibition) on cis-priming by TP and RT domain sequences provided in trans. Protein priming was further shown to induce RT conformational changes that resulted in TP-RT domain dissociation, altered priming site selection, and a gain of sensitivity to a pyrophosphate analog inhibitor. trans-priming, trans-inhibition, and trans-complementation, which requires separate TP and RT domains to reconstitute a functional RT protein, were employed to define the sequences in the TP and RT domains that could mediate physical or functional inter-and intradomain interactions. These results provide new insights into TP-RT domain interactions and conformational dynamics during protein priming and suggest novel means to inhibit protein priming by targeting these interactions and the associated conformational transitions. T he hepatitis B virus (HBV) is a major human pathogen that chronically infects over 350 million people worldwide (15, 32).Chronic HBV infection is a major cause of end-stage liver diseases, including cirrhosis and hepatocellular carcinoma, resulting in a million fatalities annually. HBV is a member of the Hepadnaviridae family, which also includes related viruses that infect other mammalian and avian species (39). In particular, the duck hepatitis B virus (DHBV) has been a widely used model to study many different aspects of HBV replication and pathogenesis. All hepadnaviruses, as pararetroviruses, replicate a short (ca. 3-kb), partially double-stranded (DS), relaxed circular DNA genome via packaging and reverse transcription of a pregenomic RNA (pgRNA) by a virally encoded reverse transcriptase (RT) (37,39,41).The hepadnavirus RT is a multifunctional protein with unique structural and functional properties (20,21). Like its retroviral counterparts, RT catalyzes the synthesis of the DS viral DNA, first the minus strand from the pgRNA template and then the plus strand from the minus-strand DNA template (10,12,35,47). Also in common with retroviral RTs, the hepadnavirus RT has an RNase H activity that degrades the pgRNA template during the synthesis of the viral minus-strand DNA (10,11,35). Thus, the central and C-terminal regions of RT harbor, respectively, the RT and RNase H domains that are homologous to retroviral RTs. Uniquely, however, the hepadnavi...

CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4-SHP2-ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple-negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine-based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2-ERK, 3) induces SHP2-dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2-ERK and driving SHP2-dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4. These studies were supported by NIH grant GM-097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund. Citation Format: Lili T. Belcastro, Anastasia Jancina, Christina Adams, Ryan D. Paulukinas, Catherine C. Moore. Leveraging a novel ITIM motif in GPCRs for targeted antibody design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 360. doi:10.1158/1538-7445.AM2017-360

PROCEEDINGS OF THE BIOCHEMICAL SOCIETY sharply, the increase corresponding to the decline in radioactivity in the acid-soluble fraction. Taken in conjunction with the results described by Billing, Barbiroli & Smellie (1969), these results suggest that there are no major changes in the rate of synthesis of RNA in rat uterus until at least 6 hr. after hormone treatment and that thereafter there is a sharp increase in the rate of synthesis of RNA. These findings are consistent with the observation that the amount of RNA in rat uterus only begins to increase about 7 hr. after oestradiol treatment.