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.
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 ( i mmmunoreceptor t yrosine‐based i nhibitory consensus m otifs), 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. Support or Funding Information These studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
Abstract 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
ABSTRACT 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.
ABSTRACT 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.
Abstract ID 95428Poster Board 248 CXCR4, a chemokine GPCR, is essential for migration of neuronal and hematopoietic cells during embryonic development, and for migration of breast cancer cells during metastasis whereby CXCR4 dysregulation promotes cell motility and invasion. Following uniform SDF stimulation, CXCR4 is rapidly phosphorylated on serine and threonine residues in the C-terminal tail, which initiates b-arrestin recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF however, significantly delays this 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 (TNBC), whereby it transduces mitogenic and migratory signals driving hyper-proliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immunoreceptor tyrosine-based inhibitory consensus motifs), a hallmark of inhibitory immune receptors with little evidence for their presence in GPCRs. Here we identify a novel ITIM motif in the chemokine GPCR CXCR4, that drives gradient signaling to SHP2 and directional motility. Specifically, first we show that gradient SDF stimulation of the CXCR4 receptor 1) delays receptor phosphorylation and trafficking, and 2) sustains signaling to a novel CXCR4-SHP2-ERK pathway. Next, we identify an ITIM motif in the CXCR4 receptor that drives gradient SDF mediated 3) sustained signaling to SHP2, and 4) directional migration in TNBC cells. Additionally, we demonstrate that this novel ITIM motif can be 5) leveraged for targeted antibody design, and 6) peptide inhibitor development. Overall 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, a CXCR4 ITIM motif is critical for transducing this SDF gradient sensing to SHP2 binding and signaling, and directional motility. Coupled with our ITIM targeted antibody and peptide inhibitor development, these data have therapeutic implications for metastatic diseases driven by aberrant CXCR4 and SHP2 expression.
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.
