The crystal structure of vaccinia virus protein E2 and perspectives on the prediction of novel viral protein folds

The morphogenesis of vaccinia virus (VACV, family Poxviridae), the smallpox vaccine, is a complex process involving multiple distinct cellular membranes and resulting in multiple different forms of infectious virion. Efficient release of enveloped virions, which promote systemic spread of infection within hosts, requires the VACV protein E2 but the molecular basis of E2 function remains unclear and E2 lacks sequence homology to any well-characterised family of proteins. We solved the crystal structure of VACV E2 to 2.3 Å resolution, revealing that it comprises two domains with novel folds: an N-terminal annular (ring) domain and a C-terminal globular (head) domain. The C-terminal head domain displays weak structural homology with cellular (pseudo)kinases but lacks conserved surface residues or kinase features, suggesting that it is not enzymatically active, and possesses a large surface basic patch that might interact with phosphoinositide lipid headgroups. Recent deep learning methods have revolutionised our ability to predict the three-dimensional structures of proteins from primary sequence alone. VACV E2 is an exemplar ‘difficult’ viral protein target for structure prediction, being comprised of multiple novel domains and lacking sequence homologues outside Poxviridae. AlphaFold2 nonetheless succeeds in predicting the structures of the head and ring domains with high and moderate accuracy, respectively, allowing accurate inference of multiple structural properties. The advent of highly accurate virus structure prediction marks a step-change in structural virology and beckons a new era of structurally-informed molecular virology.

Multi-omics reveals principles of gene regulation and pervasive non-productive transcription in the human cytomegalovirus genome

For decades, human cytomegalovirus (HCMV) was thought to express ≈200 viral proteins during lytic infection. In recent years, systems biology approaches uncovered hundreds of additional viral gene products and suggested thousands of viral sites of transcription initiation. Despite all available data, the molecular mechanisms of HCMV gene regulation remain poorly understood. Here, we provide a unifying model of productive HCMV gene expression employing transcription start site profiling combined with metabolic RNA labeling as well as integrative computational analysis of previously published big data. This approach defined the expression of >2,600 high confidence viral transcripts and explained the complex kinetics of viral protein expression by cumulative effects of translation of incoming virion-associated RNA, multiple transcription start sites with distinct kinetics per viral open reading frame, and differences in viral protein stability. Most importantly, we identify pervasive transcription of transient RNAs as a common feature of this large DNA virus with its human host.

Hepatitis B virus small envelope protein promotes HCC angiogenesis via ER stress signaling to upregulate VEGFA expression

Hepatocellular carcinoma (HCC) is a hypervascular tumor and accumulating evidence has indicated that stimulation of angiogenesis by HBV may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV viral protein and has a close clinical association with HCC, however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that forced expression of SHBs in HCC cells promoted xenograft tumor growth and increased the microvessel density (MVD) within the tumors. Consistently, HBsAg was also positively correlated with MVD count in HCC patients’ specimens. The conditioned media from the SHBs-transfected HCC cells increased the capillary tube formation and migration of human umbilical vein endothelial cells (HUVECs). Intriguingly, overexpression of SHBs increased VEGFA expression at both mRNA and protein levels. A higher VEGFA expression level was also observed in the xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues as compared to their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVECs migration and vessel formation. Furthermore, all the three unfolded protein response (UPR) sensors IRE1α, PERK and ATF6 associated with endoplasmic reticulum (ER) stress were found activated in the SHBs-expressing cells and correlated with VEGFA protein expression and secretion. Taken together, these results suggest an important role of SHBs in HCC angiogenesis and may highlight a potential target for preventive and therapeutic intervention of HBV-related HCC and its malignant progression. IMPORTANCE Chronic hepatitis B virus infection is one of the important risk factors for the development and progression of hepatocellular carcinoma (HCC). HCC is characteristic of hypervascularization even at early phases of the disease due to overexpression of angiogenic factors like vascular endothelial growth factor-A (VEGFA). However, a detailed mechanism in the HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV viral protein, i.e. small surface antigens (SHBs) can enhance the angiogenic capacity of HCC cells by upregulation of VEGFA expression both in vitro and in vivo . Mechanistically, SHBs induced endoplasmic reticulum (ER) stress which consequently activated unfolded protein response (UPR) signaling to increase VEGFA expression and secretion. This study suggests that SHBs plays an important pro-angiogenic role in HBV-associated HCC and may represent a potential target for anti-angiogenic therapy in the HCC.

INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation

The evasion of the Interferon response has important implications in Zika virus (ZIKV) disease. Mutations in ZIKV viral protein NS4B, associated with modulation of the interferon (IFN) system, have been linked to increased pathogenicity in animal models. In this study, we unravel ZIKV NS4B as antagonist of the IFN signaling cascade. Firstly, we reported the genomic characterization of NS4B isolated from a strain of the 2016 outbreak, ZIKV Brazil/2016/INMI1, and we predicted its membrane topology. Secondly, we analyzed its phylogenetic correlation with other flaviviruses, finding a high similarity with dengue virus 2 (DEN2) strains; in particular, the highest conservation was found when NS4B was aligned with the IFN inhibitory domain of DEN2 NS4B. Hence, we asked whether ZIKV NS4B was also able to inhibit the IFN signaling cascade, as reported for DEN2 NS4B. Our results showed that ZIKV NS4B was able to strongly inhibit the IFN stimulated response element and the IFN-γ-activated site transcription, blocking IFN-I/-II responses. mRNA expression levels of the IFN stimulated genes ISG15 and OAS1 were also strongly reduced in presence of NS4B. We found that the viral protein was acting by suppressing the STAT1 phosphorylation and consequently blocking the nuclear transport of both STAT1 and STAT2.

Quantification of Type I Interferon Inhibition by Viral Proteins: Ebola Virus as a Case Study

Type I interferons (IFNs) are cytokines with both antiviral properties and protective roles in innate immune responses to viral infection. They induce an antiviral cellular state and link innate and adaptive immune responses. Yet, viruses have evolved different strategies to inhibit such host responses. One of them is the existence of viral proteins which subvert type I IFN responses to allow quick and successful viral replication, thus, sustaining the infection within a host. We propose mathematical models to characterise the intra-cellular mechanisms involved in viral protein antagonism of type I IFN responses, and compare three different molecular inhibition strategies. We study the Ebola viral protein, VP35, with this mathematical approach. Approximate Bayesian computation sequential Monte Carlo, together with experimental data and the mathematical models proposed, are used to perform model calibration, as well as model selection of the different hypotheses considered. Finally, we assess if model parameters are identifiable and discuss how such identifiability can be improved with new experimental data.

Genomic and in silico Structural Characterization of Dobrava-Belgrade orthohantavirus Isolate from European Side of Turkey

Orthohantaviruses are transmitted to humans mostly through small mammals that are the reservoirs of these viruses. Because orthohantaviruses show high genetic variability through geographic regions, the genetic characterization of these viruses with whole genome sequencing is of great importance to clarify the molecular epidemiology and track their genetic changes in the reservoir hosts. We have previously reported the presence of Dobrava-Belgrade orthohantavirus (DOBV) in the Igneada region, Kirklareli province by showing antibodies against the virus in rodents and by sequencing partial genomes of the virus. Here we report the whole genome sequencing of DOBV Igneada strain directly from Apodemus flavicollis’ lung tissue by next-generation sequencing followed by phylogenetic analyses. In addition, viral protein structures of DOBV Igneada strain were modelled, and in silico prediction analyses of amino acid changes on viral protein function and stability were performed.The phylogenetic analysis showed a close relation between the DOBV Igneada strain from Turkey and DOBV Ano-Poroia strain from Greece. Similarity plot analysis revealed also similarities between DOBV Igneada strain and other DOBV strains from the Balkans such as Greece, Croatia, and Slovenia. Additionally, in silico prediction suggested that G318E, Y322H, and S324P mutations on Gn glycoprotein are deleterious, and all amino acid changes decrease the stability of both Gn and Gc glycoproteins.In conclusion, full orthohantaviral genomes can be obtained directly from rodent lung tissues allowing detailed genetic and structural analyses of orthohantaviruses. The DOBV Igneada strain shows great similarity to the prototype Ano-Poroia strain, yet it was predicted that DOBV Igneada strain may have some changes on its pathogenicity and its structure warranting further research.