Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient’s respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus’s dissemination.

The D614G mutation in the Spike protein of the SARS-CoV-2 has effectively replaced the early pandemic-causing variant. Using pseudotyped lentivectors, we confirmed that the aspartate replacement by glycine in position 614 is markedly more infectious. Molecular modelling suggests that the G614 mutation facilitates transition towards an open state of the Spike protein. To explain the epidemiological success of D614G, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, the exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase, is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2. However, we did not observe a significant correlation between reported COVID-19 mortality in different countries and the prevalence of the Wuhan versus G/L variant. Nevertheless, when comparing the speed of emergence and the ultimate predominance in individual countries, it is clear that the G/L variant displays major epidemiological supremacy over the original variant.

The D614G mutation of the Spike protein is thought to be relevant for SARS-CoV-2 infection. Here we report biological and epidemiological aspects of this mutation. Using pseudotyped lentivectors, we were able to confirm that the G614 variant of the Spike protein is markedly more infectious than the ancestral D614 variant. We demonstrate by molecular modelling that the replacement of aspartate by glycine in position 614 facilitates the transition towards an open state of the Spike protein. To understand whether the increased infectivity of the D614 variant explains its epidemiological success, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. There was no significant correlation between reported COVID mortality in different countries and the prevalence of the Wuhan versus G/L variant. However, when comparing the speed of emergence and the ultimate predominance in individual countries, the G/L variant displays major epidemiological supremacy. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase (RdRp), is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2.

Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)8 having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)8 peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)8 caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)8 may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.