Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges.

Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme viral loads, which may reach 1010 virions per mL serum, hepatitis B viruses are of low abundance and productivity in individual cells. Imaging of the infections in cells is thus a particular challenge especially for cccDNA that exists only in a few copies. The review describes the significance of microscopical approaches on genome and transcript detection for understanding hepatitis B virus infections, implications for understanding treatment outcomes, and recent microscopical approaches, which have not been applied in HBV research.

Absence of Resistance Mutations in the Integrase Coding Region among ART-Experienced Patients in the Republic of the Congo.

BACKGROUND: HIV infects around one hundred thousand patients in the Republic of the Congo. Approximately 25% of them receive an antiretroviral treatment; current first-line regimens include two NRTIs and one NNRTI, reverse transcriptase inhibitors. Recently, protease inhibitors (PIs) were also introduced as second-line therapy upon clinical signs of treatment failure. Due to the limited number of molecular characterizations and amount of drug resistance data available in the Republic of the Congo, this study aims to evaluate the prevalence of circulating resistance mutations within the pol region. METHODS: HIV-positive, ART-experienced patients have been enrolled in four semi-urban localities in the Republic of the Congo. Plasma samples were collected, and viral RNA was extracted. The viral load for each patient was evaluated by RT-qPCR, following the general diagnostic procedures of the University Hospital of Bordeaux. Finally, drug resistance genotyping and phylogenetic analysis were conducted following Sanger sequencing of the pol region. RESULTS: A high diversity of HIV-1 strains was observed with many recombinant forms. Drug resistance mutations in RT and PR genes were determined and correlated to HAART. Because integrase inhibitors are rarely included in treatments in the Republic of the Congo, the prevalence of integrase drug resistance mutations before treatment was also determined. Interestingly, very few mutations were observed. CONCLUSIONS: We confirmed a high diversity of HIV-1 in the Republic of the Congo. Most patients presented an accumulation of mutations conferring resistance against NRTIs, NNRTIs and PIs. Nonetheless, the absence of integrase mutations associated with drug resistance suggests that the introduction of integrase inhibitors into therapy will be highly beneficial to patients in the Republic of the Congo.

The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids.

BACKGROUND & AIMS: Hepatitis B virus (HBV) has a DNA genome but replicates within the nucleus by reverse transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are a major target for T cell-mediated immune responses. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal. METHODS: We used virion-derived capsids containing mature rcDNA (matC) and empty capsids (empC). RNA-containing capsids (rnaC) were used as a control. The investigations comprised pull-down assays for identification of cellular interaction partners, immune fluorescence microscopy for their colocalization and electron microscopy after microinjection to determine their biological significance. RESULTS: matC and empC underwent active transport through the cytoplasm towards the nucleus, while rnaC was poorly transported. We identified the dynein light chain LL1 as a functional interaction partner linking capsids to the dynein motor complex and showed that there is no compensatory transport pathway. Using capsid and dynein LL1 mutants we characterized the required domains on the capsid and LL1. CONCLUSIONS: This is the first investigation on the detailed molecular mechanism of how matC pass the cytoplasm upon infection and how empC can be actively removed from the cytoplasm into the nucleus. Considering that hepatocytes with cytoplasmic capsids are better recognized by the T cells, we hypothesize that targeting capsid DynLL1-interaction will not only block HBV infection but also stimulate elimination of infected cells. LAY SUMMARY: In this study, we identified the molecular details of HBV translocation through the cytoplasm. Our evidence offers a new drug target which could not only inhibit infection but also stimulate immune clearance of HBV infected cells.

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages.

Viruses often interfere with the DNA damage response to better replicate in their hosts. The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) protein has been reported to modulate the activity of the DNA repair structure-specific endonuclease subunit (SLX4) complex and to promote cell cycle arrest. Vpr also interferes with the base-excision repair pathway by antagonizing the uracil DNA glycosylase (Ung2) enzyme. Using an unbiased quantitative proteomic screen, we report that Vpr down-regulates helicase-like transcription factor (HLTF), a DNA translocase involved in the repair of damaged replication forks. Vpr subverts the DDB1-cullin4-associated-factor 1 (DCAF1) adaptor of the Cul4A ubiquitin ligase to trigger proteasomal degradation of HLTF. This event takes place rapidly after Vpr delivery to cells, before and independently of Vpr-mediated G2 arrest. HLTF is degraded in lymphocytic cells and macrophages infected with Vpr-expressing HIV-1. Our results reveal a previously unidentified strategy for HIV-1 to antagonize DNA repair in host cells.

Intracellular transport and egress of hepatitis B virus.

Hepatitis B virus (HBV) replicates its genomic information in the nucleus via transcription and therefore has to deliver its partially double stranded DNA genome into the nucleus. Like other viruses with a nuclear replication phase, HBV genomes are transported inside the viral capsids first through the cytoplasm towards the nuclear envelope. Following the arrival at the nuclear pore, the capsids are transported through, using classical cellular nuclear import pathways. The arrest of nuclear import at the nucleoplasmic side of the nuclear pore is unique, however, and is where the capsids efficiently disassemble leading to genome release. In the latter phase of the infection, newly formed nucleocapsids in the cytosol have to move to budding sites at intracellular membranes carrying the three viral envelope proteins. Capsids containing single stranded nucleic acid are not enveloped, in contrast to empty and double stranded DNA containing capsids. A small linear domain in the large envelope protein and two areas on the capsid surface have been mapped, where point mutations strongly block nucleocapsid envelopment. It is possible that these domains are involved in the envelope--with capsid interactions driving the budding process. Like other enveloped viruses, HBV also uses the cellular endosomal sorting complexes required for transport (ESCRT) machinery for catalyzing budding through the membrane and away from the cytosol.

How SLX4 cuts through the mystery of HIV-1 Vpr-mediated cell cycle arrest.

Vpr is one of the most enigmatic viral auxiliary proteins of HIV. During the past twenty years, several activities have been ascribed to this viral protein, but one, its ability to mediate cell cycle arrest at the G2 to M transition has been the most extensively studied. Nonetheless, the genuine role of Vpr and its pathophysiological relevance in the viral life cycle have remained mysterious. Recent work by Laguette et al. (Cell 156:134-145, 2014) provides important insight into the molecular mechanism of Vpr-mediated G2 arrest. This study highlights for the first time how Vpr recruits the SLX4 endonuclease complex and how Vpr-induced inappropriate activation of this complex leads to G2 arrest. Here, we will discuss these findings in the light of previous work to show how they change the view of Vpr's mechanism of action. We will also discuss how these findings open new questions towards the understanding of the biological function of Vpr regarding innate immune sensing.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein.

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1(58-177) core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1(58-177), as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

The insertion of fluorescent proteins in a variable region of respiratory syncytial virus L polymerase results in fluorescent and functional enzymes but with reduced activities.

The respiratory syncytial virus (RSV) Large protein L is the catalytic subunit of the RNA-dependent RNA polymerase complex. Currently, no structural information is available for RSV L. Sequence alignments of L protein from human and bovine strains of RSV revealed the existence of two variable regions, VR1 and VR2. Following comparison with morbillivirus and rhabdovirus L genes, VR2, which is located between domains V and VI, was chosen as an insertion site for sequences encoding the epitope tag HA or the fluorescent proteins eGFP and mCherry. Recombinant tagged-L proteins co-localized with RSV N and P proteins in transfected cells. These recombinant polymerases were shown to be functional using a viral minigenome system assay, their activities being reduced by ~70% compared to the unmodified L polymerase. We have also shown by site-directed mutagenesis that the GDNQ motif (residues 810-813 for the Long strain of HRSV) is essential for L activity.

1H, 13C, and 15N resonance assignment of the central domain of hRSV transcription antitermination factor M2-1.

M2-1 is an essential co-factor of the respiratory syncytial virus, an important respiratory pathogen in infants and calves. It acts as a transcription antitermination factor which enhances the processivity of the polymerase. Within the polymerase complex, M2-1 interacts with a second co-factor, the phosphoprotein P. It has been shown previously that P and RNA bind to M2-1 in a competitive manner in vitro and that these properties are related to a central domain located between residues Glu59 and Lys177. Here we report the almost complete (1)H, (13)C and (15)N assignment of a fragment of M2-1 corresponding to this region, for further structure determination and interaction studies.

Human herpesvirus 8 genotype E in patients with Kaposi sarcoma, Peru.

To determine human herpesvirus 8 (HHV-8) K1 genotypes in patients with Kaposi sarcoma (KS) from Peru, we characterized HHV-8 in 25 KS biopsy samples. Our findings of 8 A, 1 B, 14 C, and 2 E subtypes showed high HHV-8 diversity in these patients and association between E genotype and KS development.