The function of the alphaherpesvirus UL47 tegument protein has not yet been defined. Nonetheless, previous studies with transfected cells have shown that both the herpes simplex virus type 1 homologue (hUL47, or VP13/14) and the bovine herpesvirus type 1 (BHV-1) homologue (bUL47, or VP8) have the capacity to shuttle between the nucleus and the cytoplasm. Furthermore, hUL47 packaged into the virion has also been shown to bind several individual virus-specific RNA transcripts. Here, we extend these observations and show that hUL47 binds a wide range of RNA species in vitro. It has a high affinity for polyadenylated transcripts but has no apparent selectivity for virus-encoded RNA over cellular RNA. We also show that the virion population of bUL47 binds RNA in vitro. However, while purified recombinant hUL47 retains its RNA binding activity, recombinant bUL47 does not, suggesting that the BHV-1 homologue may require virus-induced modification for its activity. We identify the minimal RNA binding domain in hUL47 as a 26-residue N-terminal peptide containing an arginine-rich motif that is essential but not sufficient for optimal RNA binding, and we demonstrate that this RNA binding domain incorporates the hUL47 minimal nuclear localization signal. In addition, we show that soon after hUL47 is expressed during infection, it colocalizes in the infected cell nucleus with ICP4, the major virus transcriptional activator. Using RNA immunoprecipitations, we demonstrate that hUL47 is also bound in vivo to at least one viral transcript, the ICP0 mRNA. Taken together, these results suggest that hUL47 may play a role in RNA biogenesis in the infected cell.
The herpes simplex virus type 1 tegument protein known as VP13/14, or hUL47, localizes to the nucleus and binds RNA. Using fluorescence loss in photobleaching analysis, we show that hUL47 undergoes nucleocytoplasmic shuttling during infection. We identify the hUL47 nuclear export signal (NES) as a C-terminal 10-residue hydrophobic peptide and measure its efficiency relative to that of the classical human immunodeficiency virus type 1 Rev NES. Finally, we show that the hUL47 NES is sensitive to the inhibitor of CRM1-mediated nuclear export leptomycin B. Hence, hUL47 joins a growing list of virus-encoded RNA-binding proteins that use CRM1 to exit the nucleus.The alphaherpesvirus UL47 gene encodes a major structural protein that is assembled into the tegument of the virus particle (1,9,11,20). Although the role of UL47 has not yet been defined, herpes simplex virus type 1 (HSV-1) variants unable to express the UL47 protein (VP13/14 or hUL47) display defects at the early stages of virus growth (21,22). In addition, all the UL47 proteins encoded by HSV-1, bovine herpesvirus 1 (BHV-1), varicella-zoster virus, equine herpesvirus 1, and pseudorabies virus exhibit a nuclear localization, suggesting a role for these proteins in that compartment of the infected cell (3,4,9,(16)(17)(18)23). An important characteristic of the UL47 proteins encoded by HSV-1 (hUL47) and BHV-1 (bUL47) is that they both undergo nucleocytoplasmic trafficking when expressed alone (4,17,18,23). We and others previously identified nonclassical arginine-rich nuclear localization signals (NLSs) in the N termini of hUL47 and bUL47 (see Fig. 2A) (17, 23), and we went on to show that the NLS of hUL47 also functions as an RNA binding domain (5). In the case of bUL47, a leucine-rich nuclear export signal (NES) was first identified in the middle of the protein by Zheng and coworkers (see Fig. 2A, NES1) (23), suggesting that the bUL47 export signal is similar to the classical HIV-1 Rev protein NES, which uses the CRM1 receptor to exit the nucleus (6,7,15,19). Subsequently, we identified a second, and in our hands more potent, NES at the N terminus of bUL47 that contained no hydrophobic residues and was resistant to treatment with the CRM1 inhibitor leptomycin B (LMB) (see Fig. 2A, NES2) (17). The export receptor for this second NES has yet to be identified.To extend our understanding of how and why these proteins may shuttle between the nucleus and cytoplasm, we investigated the nature of hUL47 nuclear export. We first wished to determine if hUL47 shuttled between the nucleus and cytoplasm of HSV-1-infected cells. Vero cells grown on glass-bottom culture dishes (MatTek) were infected at a multiplicity of infection of 5 with recombinant HSV-1 (strain 17) expressing yellow fluorescent protein (YFP)-tagged hUL47, which was described previously and behaves as wild-type virus (3). Six hours later, at a time at which hUL47 is predominantly nuclear (as shown in our previous studies), the infected cells were examined in the temperature-controlled chamber of a Zeiss ...
In 2015, there was a large outbreak of Zika virus (ZIKV) in Brazil. Despite its relatively mild impact on healthy adults, ZIKV infection during pregnancy has been associated with severe birth defects. Currently, there is no ZIKV vaccine available, but several vaccine candidates based on the ZIKV membrane (M) and envelope (Env) structural proteins showed promising results in preclinical and clinical studies. Here, the immunogenicity and protective efficacy of a non-replicating adenoviral vector type 26 (Ad26) that encodes the ZIKV M-Env antigens (Ad26.ZIKV.M-Env) was evaluated in mice and non-human primates (NHP). Ad26.ZIKV.M-Env induced strong and durable cellular and humoral immune responses in preclinical models. Humoral responses were characterized by Env-binding and ZIKV neutralizing antibody responses while cellular responses were characterized by ZIKV reactive CD4+ and CD8+ T cells. Importantly, a single immunization with a very low dose of 4x107 vp of Ad26.ZIKV.M-Env protected mice from ZIKV challenge. In NHP, a single immunization with a typical human dose of 1x1011 vp of Ad26.ZIKV.M-Env also induced Env-binding and ZIKV neutralizing antibodies and Env and M specific cellular immune responses that associated with complete protection against viremia from ZIKV challenge as measured in plasma and other body fluids. Together these data provide the rationale to progress the Ad26.ZIKV.M-Env candidate vaccine to clinical testing.
A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.
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