The UL69 gene product of human cytomegalovirus belongs to a family of regulatory proteins conserved among all herpesviruses that have in part been characterized as posttranscriptional transactivators participating in the nuclear export of RNA. Recent experiments suggested that pUL69 also acts as a posttranscriptional activator since it was demonstrated that nucleocytoplasmic shuttling via a CRM1-independent nuclear export signal is a prerequisite for its stimulatory effect on gene expression. Based on these findings we initiated studies to investigate the role of pUL69 in mRNA export and demonstrate that pUL69 efficiently promotes the cytoplasmic accumulation of unspliced RNA. Furthermore, we show that this pUL69 activity is linked to the cellular mRNA export machinery by direct protein interaction with the highly related DEXD/H-box RNA helicases UAP56 and URH49. Particularly, we identified a 12-amino-acid domain within the N terminus of pUL69 which is required for binding to UAP56 and URH49, and we could demonstrate that UAP56 interaction and nucleocytoplasmic shuttling are both prerequisites for pUL69-mediated mRNA export. Thus, we identified a novel cellular target which provides a herpesviral regulatory protein with access to a conserved cellular transport system in order to promote nuclear export of unspliced RNA.

Human cytomegalovirus encodes a number of phosphorylation-regulated proteins, including the autophosphorylating protein kinase pUL97 and the nuclear mRNA export factor pUL69. Recently, it was reported that the kinase inhibitor roscovitine induces an intranuclear aggregation of pUL69 in infected fibroblasts. Here, we demonstrate that pUL97-specific kinase inhibitors induce a similar pUL69 aggregation. Furthermore, a direct pUL69–pUL97 interaction was demonstrated by coimmunoprecipitation analyses. Deletion mapping identified the domains required for interaction in both proteins (1–140/478–532 in pUL69 and 231–336 in pUL97). Further analysis of the immunoprecipitates by in vitro kinase assays demonstrated the phosphorylation of pUL69 by pUL97. However, catalytically inactive mutants of pUL97 and interaction-negative fragments of pUL69 were phosphorylation-negative. Moreover, an analysis of the pUL69-mediated nuclear RNA export indicated a correlation of the export efficiency with the presence of active pUL97 kinase. These data suggest a specific pUL69–pUL97 interaction and pUL97-mediated phosphorylation which influences the regulatory activities of pUL69.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell–cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike’s fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike’s fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.

The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that has counterparts in all herpesviruses. Some of these proteins have been shown to function primarily at the post-transcriptional level in promoting nuclear export of viral transcripts. Consistently, this group has reported recently that pUL69 is an RNA-binding, nucleocytoplasmic shuttling protein that facilitates the cytoplasmic accumulation of unspliced mRNA via its interaction with the cellular mRNA export factor UAP56. Evidence has been presented to suggest that some of the pUL69 homologues self-interact and function in vivo as multimers. Herein, the possibility of pUL69 self-association was examined and it has been demonstrated that pUL69 can interact with itself in vitro and in vivo in order to form high-molecular-mass complexes. The self-interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses, suggesting that multimerization is a conserved feature of this protein family.

To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero. Virus neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is non-fusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to related class III fusion proteins including Vesicular Stomatitis Virus glycoprotein G or Baculovirus gp64. To explore requirements for gB9s fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus neutralizing gB-specific monoclonal antibodies (mAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing mAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB mAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection. IMPORTANCE HCMV is a major global health concern and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence is often included as a component of intervention strategies. By generation of fusion-active gB chimeras we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB9s fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.