TDP-43 Controls HIV-1 Viral Production and Virus Infectiveness.

The transactive response DNA-binding protein (TARDBP/TDP-43) is known to stabilize the anti-HIV-1 factor, histone deacetylase 6 (HDAC6). TDP-43 has been reported to determine cell permissivity to HIV-1 fusion and infection acting on tubulin-deacetylase HDAC6. Here, we studied the functional involvement of TDP-43 in the late stages of the HIV-1 viral cycle. The overexpression of TDP-43, in virus-producing cells, stabilized HDAC6 (i.e., mRNA and protein) and triggered the autophagic clearance of HIV-1 Pr55Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55Gag and Vif proteins incorporated into virions. A nuclear localization signal (NLS)-TDP-43 mutant was not able to control HIV-1 viral production and infection. Likewise, specific TDP-43-knockdown reduced HDAC6 expression (i.e., mRNA and protein) and increased the expression level of HIV-1 Vif and Pr55Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55Gag proteins in virions and their infection capacity. Therefore, for TDP-43, the TDP-43/HDAC6 axis could be considered a key factor to control HIV-1 viral production and virus infectiveness.

HIV Infection: Shaping the Complex, Dynamic, and Interconnected Network of the Cytoskeleton.

HIV-1 has evolved a plethora of strategies to overcome the cytoskeletal barrier (i.e., actin and intermediate filaments (AFs and IFs) and microtubules (MTs)) to achieve the viral cycle. HIV-1 modifies cytoskeletal organization and dynamics by acting on associated adaptors and molecular motors to productively fuse, enter, and infect cells and then traffic to the cell surface, where virions assemble and are released to spread infection. The HIV-1 envelope (Env) initiates the cycle by binding to and signaling through its main cell surface receptors (CD4/CCR5/CXCR4) to shape the cytoskeleton for fusion pore formation, which permits viral core entry. Then, the HIV-1 capsid is transported to the nucleus associated with cytoskeleton tracks under the control of specific adaptors/molecular motors, as well as HIV-1 accessory proteins. Furthermore, HIV-1 drives the late stages of the viral cycle by regulating cytoskeleton dynamics to assure viral Pr55Gag expression and transport to the cell surface, where it assembles and buds to mature infectious virions. In this review, we therefore analyze how HIV-1 generates a cell-permissive state to infection by regulating the cytoskeleton and associated factors. Likewise, we discuss the relevance of this knowledge to understand HIV-1 infection and pathogenesis in patients and to develop therapeutic strategies to battle HIV-1.

Transactive Response DNA-Binding Protein (TARDBP/TDP-43) Regulates Cell Permissivity to HIV-1 Infection by Acting on HDAC6.

The transactive response DNA-binding protein (TARDBP/TDP-43) influences the processing of diverse transcripts, including that of histone deacetylase 6 (HDAC6). Here, we assessed TDP-43 activity in terms of regulating CD4+ T-cell permissivity to HIV-1 infection. We observed that overexpression of wt-TDP-43 increased both mRNA and protein levels of HDAC6, resulting in impaired HIV-1 infection independently of the viral envelope glycoprotein complex (Env) tropism. Consistently, using an HIV-1 Env-mediated cell-to-cell fusion model, the overexpression of TDP-43 levels negatively affected viral Env fusion capacity. Silencing of endogenous TDP-43 significantly decreased HDAC6 levels and increased the fusogenic and infection activities of the HIV-1 Env. Using pseudovirus bearing primary viral Envs from HIV-1 individuals, overexpression of wt-TDP-43 strongly reduced the infection activity of Envs from viremic non-progressors (VNP) and rapid progressors (RP) patients down to the levels of the inefficient HIV-1 Envs observed in long-term non-progressor elite controllers (LTNP-EC). On the contrary, silencing endogenous TDP-43 significantly favored the infectivity of primary Envs from VNP and RP individuals, and notably increased the infection of those from LTNP-EC. Taken together, our results indicate that TDP-43 shapes cell permissivity to HIV-1 infection, affecting viral Env fusion and infection capacities by altering the HDAC6 levels and associated tubulin-deacetylase anti-HIV-1 activity.

Membrane dynamics associated with viral infection.

Viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. These processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the ER, mitochondria, Golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. In fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. In this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. By defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease.

The HDAC6/APOBEC3G complex regulates HIV-1 infectiveness by inducing Vif autophagic degradation.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) has evolved a complex strategy to overcome the immune barriers it encounters throughout an organism thanks to its viral infectivity factor (Vif), a key protein for HIV-1 infectivity and in vivo pathogenesis. Vif interacts with and promotes "apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3G" (A3G) ubiquitination and subsequent degradation by the proteasome, thus eluding A3G restriction activity against HIV-1. RESULTS: We found that cellular histone deacetylase 6 (HDAC6) directly interacts with A3G through its C-terminal BUZ domain (residues 841-1,215) to undergo a cellular co-distribution along microtubules and cytoplasm. The HDAC6/A3G complex occurs in the absence or presence of Vif, competes for Vif-mediated A3G degradation, and accounts for A3G steady-state expression level. In fact, HDAC6 directly interacts with and promotes Vif autophagic clearance, thanks to its C-terminal BUZ domain, a process requiring the deacetylase activity of HDAC6. HDAC6 degrades Vif without affecting the core binding factor ß (CBF-ß), a Vif-associated partner reported to be key for Vif- mediated A3G degradation. Thus HDAC6 antagonizes the proviral activity of Vif/CBF-ß-associated complex by targeting Vif and stabilizing A3G. Finally, in cells producing virions, we observed a clear-cut correlation between the ability of HDAC6 to degrade Vif and to restore A3G expression, suggesting that HDAC6 controls the amount of Vif incorporated into nascent virions and the ability of HIV-1 particles of being infectious. This effect seems independent on the presence of A3G inside virions and on viral tropism. CONCLUSIONS: Our study identifies for the first time a new cellular complex, HDAC6/A3G, involved in the autophagic degradation of Vif, and suggests that HDAC6 represents a new antiviral factor capable of controlling HIV-1 infectiveness by counteracting Vif and its functions.

The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor.

Zika virus (ZIKV) infection and pathogenesis are linked to the disruption of neurogenesis, congenital Zika syndrome and microcephaly by affecting neural progenitor cells. Nonstructural protein 5 (NS5) is the largest product encoded by ZIKV-RNA and is important for replication and immune evasion. Here, we studied the potential effects of NS5 on microtubules (MTs) and autophagy flux, together with the interplay of NS5 with histone deacetylase 6 (HDAC6). Fluorescence microscopy, biochemical cell-fractionation combined with the use of HDAC6 mutants, chemical inhibitors and RNA interference indicated that NS5 accumulates in nuclear structures and strongly promotes the acetylation of MTs that aberrantly reorganize in nested structures. Similarly, NS5 accumulates the p62 protein, an autophagic-flux marker. Therefore, NS5 alters events that are under the control of the autophagic tubulin-deacetylase HDAC6. HDAC6 appears to degrade NS5 by autophagy in a deacetylase- and BUZ domain-dependent manner and to control the cytoplasmic expression of NS5. Moreover, NS5 inhibits RNA-mediated RIG-I interferon (IFN) production, resulting in greater activity when autophagy is inhibited (i.e., effect correlated with NS5 stability). Therefore, it is conceivable that NS5 contributes to cell toxicity and pathogenesis, evading the IFN-immune response by overcoming HDAC6 functions. HDAC6 has emerged as an anti-ZIKV factor by targeting NS5.

Gelsolin activity controls efficient early HIV-1 infection.

BACKGROUND: HIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events. RESULTS: Here we show that gelsolin restructures cortical F-actin during HIV-1 Env-gp120-mediated signalling, without affecting cell-surface expression of receptors or viral co-receptor signalling. Remarkably, efficient HIV-1 Env-mediated membrane fusion and infection of permissive lymphocytes were impaired when gelsolin was either overexpressed or silenced, which led to a loss or gain of cortical actin, respectively. Indeed, HIV-1 Env-gp120-induced F-actin reorganization and viral receptor capping were impaired under these experimental conditions. Moreover, gelsolin knockdown promoted HIV-1 Env-gp120-mediated aberrant pseudopodia formation. These perturbed-actin events are responsible for the inhibition of early HIV-1 infection. CONCLUSIONS: For the first time we provide evidence that through its severing of cortical actin, and by controlling the amount of actin available for reorganization during HIV-1 Env-mediated viral fusion, entry and infection, gelsolin can constitute a barrier that restricts HIV-1 infection of CD4+ lymphocytes in a pre-fusion step. These findings provide important insights into the complex molecular and actin-associated dynamics events that underlie early viral infection. Thus, we propose that gelsolin is a new factor that can limit HIV-1 infection acting at a pre-fusion step, and accordingly, cell-signals that regulate gelsolin expression and/or its actin-severing activity may be crucial to combat HIV-1 infection.

Reinfection rate and disease severity of the BA.5 Omicron SARS-CoV-2 lineage compared to previously circulating variants of concern in the Canary Islands (Spain).

ABSTRACTThe emergence of the Omicron SARS-CoV-2 variant of concern has changed the COVID-19 scenario as this variant is characterized by high transmissibility and immune evasion ability. To evaluate the impact of this variant on the Canary Islands (Spain) population, we determined the reinfection rates and disease severity associated with the Omicron sublineages and the previously circulating variants of concern. We performed a retrospective observational study on 21,745 SARS-CoV-2 viral genomes collected from December 2020 to July 2022 in the Canary Islands (Spain). We compared the reinfection rates between lineages using pairwise proportion and Fisher's exact tests. To assess disease severity, we studied the association of Alpha, Delta, BA.1, BA.2, BA.5, and other risk factors on 28-day hospital mortality using logistic regression and Cox proportional hazard models. We observed 127 bona fide reinfection cases throughout the study period. We found that BA.5 had the highest reinfection rate compared to other lineages (vs. Delta p = 2.89 × 10-25; vs. BA.1 p = 5.17 × 10-11; vs. BA.2 p = 0.002). Among the 1,094 hospitalized patients, multivariate logistic regression showed that Alpha (Odds Ratio [OR] = 0.45, 95% Confidence Interval [CI] = 0.23-0.87, p = 0.02), BA.2 (OR = 0.38, 95% CI = 0.22-0.63, p = 1.91 × 10-4), and BA.5 (OR = 0.30, 95% CI = 0.16-0.55, p = 1.05 × 10-4) had lower 28-day hospital mortality compared to Delta. These results were confirmed by using Cox proportional hazard models. Omicron lineages, and in particular BA.5, were associated with higher reinfection rates and lower disease severity (28-day hospital mortality) than previously circulating variants of concern.

Bioinformatic approaches to draft the viral genome sequence of Canary Islands cases related to the multicountry mpox virus 2022-outbreak.

On July 23, 2022, monkeypox disease (mpox) was declared a Public Emergency of International Concern (PHEIC) by the World Health Organization (WHO) due to a multicountry outbreak. In Europe, several cases of mpox virus (MPXV) infection related to this outbreak were detected in the Canary Islands (Spain). Here we describe the combination of viral DNA sequencing and bioinformatic approaches, including methods for de novo genome assembly and short- and long-read technologies, used to reconstruct the first MPXV genome isolated in the Canary Islands on the 31st of May 2022 from a male adult patient with mild symptoms. The same sequencing and bioinformatic approaches were then validated with three other positive cases of MPXV infection from the same mpox outbreak. We obtained the best results using a reference-based approach with short reads, evidencing 46-79 nucleotide variants against viral sequences from the 2018-2019 mpox outbreak and placing the viral sequences in the new B.1 sublineage of clade IIb of the MPXV classification. This study of MPXV demonstrates the potential of metagenomics sequencing for rapid and precise pathogen identification.

The Characteristics of the HIV-1 Env Glycoprotein Are Linked With Viral Pathogenesis.

The understanding of HIV-1 pathogenesis and clinical progression is incomplete due to the variable contribution of host, immune, and viral factors. The involvement of viral factors has been investigated in extreme clinical phenotypes from rapid progressors to long-term non-progressors (LTNPs). Among HIV-1 proteins, the envelope glycoprotein complex (Env) has been concentrated on in many studies for its important role in the immune response and in the first steps of viral replication. In this study, we analyzed the contribution of 41 Envs from 24 patients with different clinical progression rates and viral loads (VLs), LTNP-Elite Controllers (LTNP-ECs); Viremic LTNPs (vLTNPs), and non-controller individuals contemporary to LTNPs or recent, named Old and Modern progressors. We studied the Env expression, the fusion and cell-to-cell transfer capacities, as well as viral infectivity. The sequence and phylogenetic analysis of Envs were also performed. In every functional characteristic, the Envs from subjects with viral control (LTNP-ECs and vLTNPs) showed significant lower performance compared to those from the progressor individuals (Old and Modern). Regarding sequence analysis, the variable loops of the gp120 subunit of the Env (i.e., V2, V4, and mainly V5) of the progressor individuals showed longer and more glycosylated sequences than controller subjects. Therefore, HIV-1 Envs from virus of patients presenting viremic control and the non-progressor clinical phenotype showed poor viral functions and shorter sequences, whereas functional Envs were associated with virus of patients lacking virological control and with progressor clinical phenotypes. These correlations support the role of Env genotypic and phenotypic characteristics in the in vivo HIV-1 infection and pathogenesis.

Contribution of the HIV-1 Envelope Glycoprotein to AIDS Pathogenesis and Clinical Progression.

In the absence of antiviral therapy, HIV-1 infection progresses to a wide spectrum of clinical manifestations that are the result of an entangled contribution of host, immune and viral factors. The contribution of these factors is not completely established. Several investigations have described the involvement of the immune system in the viral control. In addition, distinct HLA-B alleles, HLA-B27, -B57-58, were associated with infection control. The combination of these elements and antiviral host restriction factors results in different clinical outcomes. The role of the viral proteins in HIV-1 infection has been, however, less investigated. We will review contributions dedicated to the pathogenesis of HIV-1 infection focusing on studies identifying the function of the viral envelope glycoprotein (Env) in the clinical progression because of its essential role in the initial events of the virus life-cycle. Some analysis showed that inefficient viral Envs were dominant in non-progressor individuals. These poorly-functional viral proteins resulted in lower cellular activation, viral replication and minor viral loads. This limited viral antigenic production allows a better immune response and a lower immune exhaustion. Thus, the properties of HIV-1 Env are significant in the clinical outcome of the HIV-1 infection and AIDS pathogenesis.

Tracing the trajectories of SARS-CoV-2 variants of concern between December 2020 and September 2021 in the Canary Islands (Spain).

Several variants of concern (VOCs) explain most of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic waves in Europe. We aimed to dissect the spread of the SARS-CoV-2 VOCs in the Canary Islands (Spain) between December 2020 and September 2021 at a micro-geographical level. We sequenced the viral genome of 8,224 respiratory samples collected in the archipelago. We observed that Alpha (B.1.1.7) and Delta (B.1.617.2 and sublineages) were ubiquitously present in the islands, while Beta (B.1.351) and Gamma (P.1/P.1.1) had a heterogeneous distribution and were responsible for fewer and more controlled outbreaks. This work represents the largest effort for viral genomic surveillance in the Canary Islands so far, helping the public health bodies in decision-making throughout the pandemic.

Nanomaterials to combat SARS-CoV-2: Strategies to prevent, diagnose and treat COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the associated coronavirus disease 2019 (COVID-19), which severely affect the respiratory system and several organs and tissues, and may lead to death, have shown how science can respond when challenged by a global emergency, offering as a response a myriad of rapid technological developments. Development of vaccines at lightning speed is one of them. SARS-CoV-2 outbreaks have stressed healthcare systems, questioning patients care by using standard non-adapted therapies and diagnostic tools. In this scenario, nanotechnology has offered new tools, techniques and opportunities for prevention, for rapid, accurate and sensitive diagnosis and treatment of COVID-19. In this review, we focus on the nanotechnological applications and nano-based materials (i.e., personal protective equipment) to combat SARS-CoV-2 transmission, infection, organ damage and for the development of new tools for virosurveillance, diagnose and immune protection by mRNA and other nano-based vaccines. All the nano-based developed tools have allowed a historical, unprecedented, real time epidemiological surveillance and diagnosis of SARS-CoV-2 infection, at community and international levels. The nano-based technology has help to predict and detect how this Sarbecovirus is mutating and the severity of the associated COVID-19 disease, thereby assisting the administration and public health services to make decisions and measures for preparedness against the emerging variants of SARS-CoV-2 and severe or lethal COVID-19.

The Interplay of HIV and Autophagy in Early Infection.

HIV/AIDS is still a global threat despite the notable efforts made by the scientific and health communities to understand viral infection, to design new drugs or to improve existing ones, as well as to develop advanced therapies and vaccine designs for functional cure and viral eradication. The identification and analysis of HIV-1 positive individuals that naturally control viral replication in the absence of antiretroviral treatment has provided clues about cellular processes that could interact with viral proteins and RNA and define subsequent viral replication and clinical progression. This is the case of autophagy, a degradative process that not only maintains cell homeostasis by recycling misfolded/old cellular elements to obtain nutrients, but is also relevant in the innate and adaptive immunity against viruses, such as HIV-1. Several studies suggest that early steps of HIV-1 infection, such as virus binding to CD4 or membrane fusion, allow the virus to modulate autophagy pathways preparing cells to be permissive for viral infection. Confirming this interplay, strategies based on autophagy modulation are able to inhibit early steps of HIV-1 infection. Moreover, autophagy dysregulation in late steps of the HIV-1 replication cycle may promote autophagic cell-death of CD4+ T cells or control of HIV-1 latency, likely contributing to disease progression and HIV persistence in infected individuals. In this scenario, understanding the molecular mechanisms underlying HIV/autophagy interplay may contribute to the development of new strategies to control HIV-1 replication. Therefore, the aim of this review is to summarize the knowledge of the interplay between autophagy and the early events of HIV-1 infection, and how autophagy modulation could impair or benefit HIV-1 infection and persistence, impacting viral pathogenesis, immune control of viral replication, and clinical progression of HIV-1 infected patients.

Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling.

OBJECTIVES: Limited testing capacity has characterized the ongoing coronavirus disease 2019 (COVID-19) pandemic in Spain, hampering timely control of outbreaks and opportunities to reduce the escalation of community transmission. This study investigated the potential to use sample pooling, followed by one-step retrotranscription and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to increase testing capacity for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). METHODS: Various pool sizes (five, 10 and 15 samples) were evaluated prior to RNA extraction followed by standard RT-qPCR for the diagnosis of COVID-19. The pool size achieving reproducible results with individual sample testing was subsequently used to assess nasopharyngeal samples in a tertiary hospital in August 2020. RESULTS: A pool size of five samples had higher sensitivity compared with pool sizes of 10 and 15 samples, showing a mean cycle threshold (Ct) shift of 3.5 [standard deviation (SD) 2.2] between the pooled test and positive samples in the pool. Next, a pool size of five was used to test a total of 895 pools (4475 prospective samples) using two different RT-qPCR kits. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct shift [2.2 (SD 2.4)] between the pool and individual samples. This strategy enables detection of individual positive samples in positive pools with Ct of 16.7-39.4. CONCLUSIONS: Grouping samples into pools of five for RT-qPCR resulted in an increase in SARS-CoV-2 testing capacity with minimal loss of sensitivity compared with testing each sample individually.

Zika Virus Pathogenesis: A Battle for Immune Evasion.

Zika virus (ZIKV) infection and its associated congenital and other neurological disorders, particularly microcephaly and other fetal developmental abnormalities, constitute a World Health Organization (WHO) Zika Virus Research Agenda within the WHO's R&D Blueprint for Action to Prevent Epidemics, and continue to be a Public Health Emergency of International Concern (PHEIC) today. ZIKV pathogenicity is initiated by viral infection and propagation across multiple placental and fetal tissue barriers, and is critically strengthened by subverting host immunity. ZIKV immune evasion involves viral non-structural proteins, genomic and non-coding RNA and microRNA (miRNA) to modulate interferon (IFN) signaling and production, interfering with intracellular signal pathways and autophagy, and promoting cellular environment changes together with secretion of cellular components to escape innate and adaptive immunity and further infect privileged immune organs/tissues such as the placenta and eyes. This review includes a description of recent advances in the understanding of the mechanisms underlying ZIKV immune modulation and evasion that strongly condition viral pathogenesis, which would certainly contribute to the development of anti-ZIKV strategies, drugs, and vaccines.

A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor.

The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5' untranslated region (5'-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.

The tight junction-associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection.

The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.

PI4P5-kinase Ialpha is required for efficient HIV-1 entry and infection of T cells.

HIV-1 envelope (Env) triggers membrane fusion between the virus and the target cell. The cellular mechanism underlying this process is not well known. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is known to be important for the late steps of the HIV-1 infection cycle by promoting Gag localization to the plasma membrane during viral assembly, but it has not been implicated in early stages of HIV-1 membrane-related events. In this study, we show that binding of the initial HIV-1 Env-gp120 protein induces PIP(2) production in permissive lymphocytes through the activation of phosphatidylinositol-4-phosphate 5-kinase (PI4P5-K) Ialpha. Overexpression of wild-type PI4P5-K Ialpha increased HIV-1 Env-mediated PIP(2) production and enhanced viral replication in primary lymphocytes and CEM T cells, whereas PIP(2) production and HIV-1 infection were both severely reduced in cells overexpressing the kinase-dead mutant D227A (D/A)-PI4P5-K Ialpha. Similar results were obtained with replicative and single-cycle HIV-1 particles. HIV-1 infection was also inhibited by knockdown of endogenous expression of PI4P5-K Ialpha. These data indicate that PI4P5-K Ialpha-mediated PIP(2) production is crucial for HIV-1 entry and the early steps of infection in permissive lymphocytes.

Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection.

OBJECTIVES: The ongoing COVID-19 pandemic continues to impose demands on diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, this study assessed the sensitivity of a number of one-step retrotranscription and quantitative polymerase chain reaction (RT-qPCR) solutions to detect SARS-CoV-2. METHODS: Six different RT-qPCR alternatives were evaluated for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. The one with best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating; thus overcoming the RNA extraction step. RESULTS: A wide variability in the sensitivity of RT-qPCR solutions was found that was associated with a range of false negatives from 2% (0.3-7.9%) to 39.8% (30.2-50.2%). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0%), in the range of some of the solutions based on standard RNA extractions. CONCLUSIONS: Sensitivity limitations of currently used RT-qPCR solutions were found. These results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.