Ali Amara

Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function_. Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 Å resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection.

We recently identified the scaffolding RACK1 protein as an important host-dependency factor for dengue virus (DENV), a positive-stranded RNA virus responsible for the most prevalent mosquito-borne viral disease worldwide. Here, we have performed the first RACK1 interactome in human cells and identified Vigilin and SERBP1 as DENV host-dependency factors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus responsible for major outbreaks of disease since 2004 in the Indian Ocean islands, South east Asia, and the Americas. CHIKV causes debilitating musculoskeletal disorders in humans that are characterized by fever, rash, polyarthralgia, and myalgia. The disease is often self-limiting and nonlethal; however, some patients experience atypical or severe clinical manifestations, as well as a chronic rheumatic syndrome. Unfortunately, no efficient antivirals against CHIKV infection are available so far, highlighting the importance of deepening our knowledge of CHIKV host cell interactions and viral replication strategies. In this review, we discuss recent breakthroughs in the molecular mechanisms that regulate CHIKV infection and lay down the foundations to understand viral pathogenesis. We describe the role of the recently identified host factors co-opted by the virus for infection and pathogenesis, and emphasize the importance of CHIKV nonstructural proteins in both replication complex assembly and host immune response evasion.

Several studies have analyzed antiviral immune pathways in late-stage severe COVID-19. However, the initial steps of SARS-CoV-2 antiviral immunity are poorly understood. Here we have isolated primary SARS-CoV-2 viral strains and studied their interaction with human plasmacytoid predendritic cells (pDCs), a key player in antiviral immunity. We show that pDCs are not productively infected by SARS-CoV-2. However, they efficiently diversified into activated P1-, P2-, and P3-pDC effector subsets in response to viral stimulation. They expressed CD80, CD86, CCR7, and OX40 ligand at levels similar to influenza virus–induced activation. They rapidly produced high levels of interferon-α, interferon-λ1, IL-6, IP-10, and IL-8. All major aspects of SARS-CoV-2–induced pDC activation were inhibited by hydroxychloroquine. Mechanistically, SARS-CoV-2–induced pDC activation critically depended on IRAK4 and UNC93B1, as established using pDC from genetically deficient patients. Overall, our data indicate that human pDC are efficiently activated by SARS-CoV-2 particles and may thus contribute to type I IFN–dependent immunity against SARS-CoV-2 infection.

Several studies have analyzed antiviral immune pathways in late-stage severe COVID-19. However, the initial steps of SARS-CoV-2 antiviral immunity are poorly understood. Here, we have isolated primary SARS-CoV-2 viral strains, and studied their interaction with human plasmacytoid pre-dendritic cells (pDC), a key player in antiviral immunity. We show that pDC are not productively infected by SARS-CoV-2. However, they efficiently diversified into activated P1-, P2-, and P3-pDC effector subsets in response to viral stimulation. They expressed CD80, CD86, CCR7, and OX40 ligand at levels similar to influenza virus-induced activation. They rapidly produced high levels of interferon-α, interferon-λ1, IL-6, IP-10, and IL-8. All major aspects of SARS-CoV-2-induced pDC activation were inhibited by hydroxychloroquine. Mechanistically, SARS-CoV-2-induced pDC activation critically depended on IRAK4 and UNC93B1, as established using pDC from genetically deficient patients. Overall, our data indicate that human pDC are efficiently activated by SARS-CoV-2 particles and may thus contribute to type I IFN-dependent immunity against SARS-CoV-2 infection.

Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.

We used next-generation sequencing to evaluate the quantity and genetic diversity of the HIV envelope gene in various compartments in eight patients with acute infection. Plasma (PL) and seminal fluid (SF) were available for all patients, whole blood (WB) for seven, non-spermatozoid cells (NSC) for four, and saliva (SAL) for three. Median HIV-1 RNA was 6.2 log₁₀ copies/mL [IQR: 5.5–6.95] in PL, 4.9 log₁₀ copies/mL [IQR: 4.25–5.29] in SF, and 4.9 log₁₀ copies/mL [IQR: 4.46–5.09] in SAL. Median HIV-1 DNA was 4.1 log₁₀ copies/10⁶ PBMCs [IQR: 3.15–4.15] in WB and 2.6 log₁₀ copies /10⁶ Cells [IQR: 2.23–2.75] in NSC. The median overall diversity per patient varied from 0.0005 to 0.0232, suggesting very low diversity, confirmed by the clonal aspect of most of the phylogenetic trees. One single haplotype was present in all compartments for five patients in the earliest stage of infection. Evidence of higher diversity was established for two patients in PL and WB, suggesting compartmentalization. Our study shows low diversity of the env gene in the first stages of infection followed by the rapid establishment of cellular reservoirs of the virus. Such clonality could be exploited in the search for early patient-specific therapeutic solutions.

In view of the fast viremia decline obtained with integrase inhibitors, we studied the respective effects of initiating efavirenz (EFV) or raltegravir (RAL)-based antiretroviral therapy (ART) regimens on human immunodeficiency virus (HIV)-1 deoxyribonucleic acid (DNA) levels and inflammation biomarkers in the highly inflammatory setting of advanced HIV-1 disease with tuberculosis (TB) coinfection. We followed cell-associated HIV-1 DNA, high-sensitivity C-reactive protein (hsCRP), interleukin 6 (IL-6), soluble CD14 and D-Dimer levels for 48 weeks after ART initiation in the participants to the ANRS12-180 REFLATE-TB study. This phase II open-label randomized study included ART-naive people with HIV and TB treated with rifampicin to receive RAL 400 mg twice daily (RAL400), RAL 800 mg twice daily (RAL800) or EFV 600 mg QD with tenofovir and lamivudine. In 146 participants, the median (interquartile range [IQR]) week (W)0 HIV-1 DNA level was 4.7 (IQR, 4.3–5.1) log₁₀ copies/10⁶ CD4⁺, and the reduction by W48 was −0.8 log₁₀ copies/10⁶ CD4⁺ on EFV, −0.9 on RAL400, and −1.0 on RAL800 (P = .74). Baseline median (IQR) hsCRP, IL-6, sCD14, and D-Dimer levels were 6.9 (IQR, 3.3–15.6) mg/L, 7.3 (IQR, 3.5–12.3) pg/mL, 3221 (IQR, 2383–4130) ng/mL, and 975 (IQR, 535–1970) ng/mL. All biomarker levels decreased over the study: the overall W0–W48 mean (95% confidence interval) fold-change on ART was 0.37 (IQR, 0.28–0.48) for hsCRP, 0.42 (IQR, 0.35–0.51) for IL-6, 0.51 (IQR, 0.47–0.56) for sCD14, and 0.39 (IQR, 0.32–0.47) for D-Dimers. There were no differences in biomarker reduction across treatment arms. In participants with HIV and TB, EFV, RAL400, or RAL800 effectively and equally reduced inflammation and HIV-1 DNA levels.