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During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5′-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.

BACKGROUND: The ongoing COVID-19 pandemic has had a significant impact worldwide, with an incommensurable social and economic burden. The rapid development of safe and protective vaccines against this disease is a global priority. CoronaVac is a vaccine prototype based on inactivated SARS-CoV-2, which has shown promising safety and immunogenicity profiles in pre-clinical studies and phase 1/2 trials in China. To this day, four phase 3 clinical trials are ongoing with CoronaVac in Brazil, Indonesia, Turkey, and Chile. This article reports the safety and immunogenicity results obtained in a subgroup of participants aged 18 years and older enrolled in the phase 3 Clinical Trial held in Chile. METHODS: This is a multicenter phase 3 clinical trial. Healthcare workers aged 18 years and older were randomly assigned to receive two doses of CoronaVac or placebo separated by two weeks (0-14). We report preliminary safety results obtained for a subset of 434 participants, and antibody and cell-mediated immunity results obtained in a subset of participants assigned to the immunogenicity arm. The primary and secondary aims of the study include the evaluation of safety parameters and immunogenicity against SARS-CoV-2 after immunization, respectively. This trial is registered at clinicaltrials.gov ( NCT04651790 ). FINDINGS: The recruitment of participants occurred between November 27 (th) , 2020, until January 9 (th) , 2021. 434 participants were enrolled, 397 were 18-59 years old, and 37 were ≥60 years old. Of these, 270 were immunized with CoronaVac, and the remaining 164 participants were inoculated with the corresponding placebo. The primary adverse reaction was pain at the injection site, with a higher incidence in the vaccine arm (55.6%) than in the placebo arm (40.0%). Moreover, the incidence of pain at the injection site in the 18-59 years old group was 58.4% as compared to 32.0% in the ≥60 years old group. The seroconversion rate for specific anti-S1-RBD IgG was 47.8% for the 18-59 years old group 14 days post immunization (p.i.) and 95.6% 28 and 42 days p.i. For the ≥60 years old group, the seroconversion rate was 18.1%, 100%, and 87.5% at 14, 28, and 42 days p.i., respectively. Importantly, we observed a 95.7% seroconversion rate in neutralizing antibodies for the 18-59 years old group 28 and 42 days p.i. The ≥60 years old group exhibited seroconversion rates of 90.0% and 100% at 28 and 42 days p.i. Interestingly, we did not observe a significant seroconversion rate of anti-N-SARS-CoV-2 IgG for the 18-59 years old group. For the participants ≥60 years old, a modest rate of seroconversion at 42 days p.i. was observed (37.5%). We observed a significant induction of a T cell response characterized by the secretion of IFN-γ upon stimulation with Mega Pools of peptides derived from SARS-CoV-2 proteins. No significant differences between the two age groups were observed for cell-mediated immunity. INTERPRETATION: Immunization with CoronaVac in a 0-14 schedule in adults of 18 years and older in the Chilean population is safe and induces specific IgG production against the S1-RBD with neutralizing capacity, as well as the activation of T cells secreting IFN-γ, upon recognition of SARS-CoV-2 antigens. FUNDING: Ministry of Health of the Chilean Government; Confederation of Production and Commerce, Chile; Consortium of Universities for Vaccines and Therapies against COVID-19, Chile; Millennium Institute on Immunology and Immunotherapy.

To determine the impact of a booster dose on the humoral response in individuals inoculated with a complete schedule of any SARS-CoV-2 vaccine, we evaluated the neutralizing antibody (NAb) titres of homologous or heterologous booster doses over a 90-days period in CoronaVac vaccinees from 3 centres in Santiago, Chile.Individuals previously inoculated with 2 doses of CoronaVac (N = 523) were recruited in the context of the REFUERZO clinical trial (NCT04992182) and received either placebo (N = 129), or a booster dose of CoronaVac (N = 134), BNT162b2 (N = 133), or ChAdOx1 (N = 127). Pseudovirus neutralizing antibody titres (pVNT) were determined at baseline (day 0) as well as at days 14, 30, 60, and 90 after booster dose administration.Inoculating a booster dose increases the pVNTs titres at days 14 and 30 in all groups, (13.5- and 12.0-fold increase for the CoronaVac group; 247.0- and 212.3-fold increase for the BTN162b2 group; and 89.1- and 128.1-fold increase for ChAdOx1 at each time point, respectively) with a decline observed at days 60 and 90. However, although pVNTs remained significantly higher for the BTN162b2 and ChAdOx1 groups at days 60 and 90, NAb titres reached baseline levels in the CoronaVac group at 90 days after inoculation.A single heterologous booster (BTN162b2 or ChAdOx1) in individuals who completed the CoronaVac primary series resulted in an important increase in NAb titres remaining significantly higher at least for 90 days. These data may directly impact middle- and low-income countries currently using CoronaVac as the main vaccination strategy.

BackgroundThe ongoing COVID-19 pandemic has had a significant impact worldwide, with an incommensurable social and economic burden. The rapid development of safe and protective vaccines against this disease is a global priority. CoronaVac is a vaccine prototype based on inactivated SARS-CoV-2, which has shown promising safety and immunogenicity profiles in pre-clinical studies and phase 1/2 trials in China. To this day, four phase 3 clinical trials are ongoing with CoronaVac in Brazil, Indonesia, Turkey, and Chile. This article reports the safety and immunogenicity results obtained in a subgroup of participants aged 18 years and older enrolled in the phase 3 Clinical Trial held in Chile.MethodsThis is a multicenter phase 3 clinical trial. Healthcare workers aged 18 years and older were randomly assigned to receive two doses of CoronaVac or placebo separated by two weeks (0-14). We report preliminary safety results obtained for a subset of 434 participants, and antibody and cell-mediated immunity results obtained in a subset of participants assigned to the immunogenicity arm. The primary and secondary aims of the study include the evaluation of safety parameters and immunogenicity against SARS-CoV-2 after immunization, respectively. This trial is registered at clinicaltrials.gov (NCT04651790).FindingsThe recruitment of participants occurred between November 27th, 2020, until January 9th, 2021. 434 participants were enrolled, 397 were 18-59 years old, and 37 were ≥60 years old. Of these, 270 were immunized with CoronaVac, and the remaining 164 participants were inoculated with the corresponding placebo. The primary adverse reaction was pain at the injection site, with a higher incidence in the vaccine arm (55.6%) than in the placebo arm (40.0%). Moreover, the incidence of pain at the injection site in the 18-59 years old group was 58.4% as compared to 32.0% in the ≥60 years old group. The seroconversion rate for specific anti-S1-RBD IgG was 47.8% for the 18-59 years old group 14 days post immunization (p.i.) and 95.6% 28 and 42 days p.i. For the ≥60 years old group, the seroconversion rate was 18.1%, 100%, and 87.5% at 14, 28, and 42 days p.i., respectively. Importantly, we observed a 95.7% seroconversion rate in neutralizing antibodies for the 18-59 years old group 28 and 42 days p.i. The ≥60 years old group exhibited seroconversion rates of 90.0% and 100% at 28 and 42 days p.i. Interestingly, we did not observe a significant seroconversion rate of anti-N-SARS-CoV-2 IgG for the 18-59 years old group. For the participants ≥60 years old, a modest rate of seroconversion at 42 days p.i. was observed (37.5%). We observed a significant induction of a T cell response characterized by the secretion of IFN-γ upon stimulation with Mega Pools of peptides derived from SARS-CoV-2 proteins. No significant differences between the two age groups were observed for cell-mediated immunity.InterpretationImmunization with CoronaVac in a 0-14 schedule in adults of 18 years and older in the Chilean population is safe and induces specific IgG production against the S1-RBD with neutralizing capacity, as well as the activation of T cells secreting IFN-γ, upon recognition of SARS-CoV-2 antigens.FundingMinistry of Health of the Chilean Government; Confederation of Production and Commerce, Chile; Consortium of Universities for Vaccines and Therapies against COVID-19, Chile; Millennium Institute on Immunology and Immunotherapy.

BackgroundCoronaVac®is an inactivated SARS-CoV-2 vaccine approved by the World Health Organization. Previous studies reported increased levels of neutralizing antibodies and specific T cells two- and four-weeks after two doses of CoronaVac®, but the levels of neutralizing antibodies are reduced at six to eight months after two doses. Here we report the effect of a booster dose of CoronaVac®on the anti-SARS-CoV-2 immune response generated against variants of concern (VOC) Delta and Omicron in adults participating in a phase 3 clinical trial in Chile.MethodsVolunteers immunized with two doses of CoronaVac®in a four-week interval received a booster dose of the same vaccine between twenty-four and thirty weeks after the 2nd dose. Four weeks after the booster dose, neutralizing antibodies and T cell responses were measured. Neutralization capacities and T cell activation against VOC Delta and Omicron were detected at four weeks after the booster dose.FindingsWe observed a significant increase in neutralizing antibodies at four weeks after the booster dose. We also observed an increase in CD4+T cells numbers over time, reaching a peak at four weeks after the booster dose. Furthermore, neutralizing antibodies and SARS-CoV-2 specific T cells induced by the booster showed activity against VOC Delta and Omicron.InterpretationOur results show that a booster dose of CoronaVac®increases the anti-SARS-CoV-2 humoral and cellular immune responses in adults. Immunity induced by a booster dose of CoronaVac®is active against VOC, suggesting an effective protection.

We report neutralizing antibody titers (NAbTs) elicited by CoronaVac and BNT162b2 vaccines in healthcare workers with and without prior SARS-CoV-2 infection using both a pseudotype-based assay and a commercial kit. NAbTs were higher for the mRNA vaccine and increased in all previously infected. Good correlation between both assays was found.

N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and several viral RNA including human respiratory syncytial virus (HRSV). Here, we evaluated the impact of m6A writers, erasers and readers on HRSV genomic RNA accumulation and inclusion bodies assembly during viral replication. We observed that the METTL3/METTL14 m6A writer complex plays a negative role in HRSV protein synthesis and viral titers, while m6A erasers FTO and ALKBH5 had the opposite effect. We also observed that m6A readers YTHDF1-3 bind to the viral genomic RNA inducing a decrease in its intracellular levels and thus, inhibiting viral replication. Finally, we observed that overexpression of YTHDFs proteins caused a decrease in the size of inclusion bodies (IBs), accompanied by an increase in their number. METTL3 knockdown cells showed an opposite effect indicating that the dynamics of IBs assembly and coalescence are strongly affected by m6A readers in a mechanism dependent on m6A writers. Taken together, our results demonstrated that the m6A modification negatively affects HRSV replication, possibly through a mechanism involving the assembly of inclusion bodies, the main factories of viral genomic RNA synthesis.

An HIV-1–based pseudotype allows the rapid quantification of neutralizing antibody titers against the SARS-CoV-2 spike protein., Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19–related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.

The mechanisms involved in the posttranscriptional control of the replicative cycle of the human immunodeficiency virus (HIV), specifically the molecular events which allow the interaction between the viral genomic RNA (gRNA) and the cellular machinery for the transport, translation, or intracellular packaging, have not been yet elucidated. In this chapter, we describe the in situ hybridization-proximity ligation assay (ISH-PLA) to characterize interactions between the genomic RNA (gRNA) of HIV-1 and viral proteins or host proteins involved in nuclear export and translation initiation. We also present data that validate the ISH-PLA as a simple and useful tool to study HIV-1 gRNA-protein interactions within cells.

The mechanisms involved in the posttranscriptional control of the replicative cycle of the human immunodeficiency virus (HIV), specifically the molecular events which allow the interaction between the viral genomic RNA (gRNA) and the cellular machinery for the transport, translation, or intracellular packaging, have not been yet elucidated. In this chapter, we describe the in situ hybridization-proximity ligation assay (ISH-PLA) to characterize interactions between the genomic RNA (gRNA) of HIV-1 and viral proteins or host proteins involved in nuclear export and translation initiation. We also present data that validate the ISH-PLA as a simple and useful tool to study HIV-1 gRNA-protein interactions within cells.

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) unspliced mRNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the unspliced mRNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF restricts HIV-1 replication by interfering with Gag synthesis from the unspliced mRNA. Our results indicate that CTIF associates with Rev through its N-terminal domain and is recruited onto the unspliced mRNA ribonucleoprotein complex in order to block translation. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that CTIF restricts HIV-2 but not MLV Gag synthesis indicating an inhibitory mechanism conserved in Rev-expressing human lentiviruses.

Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with Rev to promote Gag synthesis. Interestingly, molecular docking analyses revealed the assembly of a Rev-CBP80-eIF4AI complex that is organized around the Rev response element (RRE). Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.