Mother's smoking habits affects IL10 methylation but not asthma in Ecuadorian children.

There is no evidence evaluating the IL10 epigenetic upregulation among mestizo children in a high-altitude Andean city in Latin America. OBJECTIVE: To identify polymorphisms and methylation profiles in the IL10 gene associated with asthma in children aged 5 to 11. METHODS: A case-control study was conducted with asthmatic and non-asthmatic children aged 5 to 11 years in Cuenca-Ecuador. Data on allergic diseases and risk factors were collected through a questionnaire for parents. Atopy was measured by skin prick test (SPT) to relevant aeroallergens. Three IL10 single nucleotide polymorphisms were evaluated in all participants, and methylation analysis was performed in 54 participants. Association between risk factors, allergic diseases and genetic factors were estimated using multivariate logistic regression. RESULTS: The results of polymorphisms showed no differences between cases and controls when comparing the SNPs rs3024495, rs3024496, rs1800896 allelic and genotypic frequencies. In the methylation analysis, no differences in the IL10 methylation profile were found between cases and controls; however, the multivariate analysis showed an association between the mother's smoking habits and the IL10 methylation profile. CONCLUSION: Smoking habit could be essential as an environmental exposure factor in regulating gene expression in children with asthma.

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

hACE2-Induced Allosteric Activation in SARS-CoV versus SARS-CoV-2 Spike Assemblies Revealed by Structural Dynamics.

SARS-CoV and SARS-CoV-2 cell entry begins when spike glycoprotein (S) docks with the human ACE2 (hACE2) receptor. While the two coronaviruses share a common receptor and architecture of S, they exhibit differences in interactions with hACE2 as well as differences in proteolytic processing of S that trigger the fusion machine. Understanding how those differences impact S activation is key to understand its function and viral pathogenesis. Here, we investigate hACE2-induced activation in SARS-CoV and SARS-CoV-2 S using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS revealed differences in dynamics in unbound S, including open/closed conformational switching and D614G-induced S stability. Upon hACE2 binding, notable differences in transduction of allosteric changes were observed extending from the receptor binding domain to regions proximal to proteolytic cleavage sites and the fusion peptide. Furthermore, we report that dimeric hACE2, the native oligomeric form of the receptor, does not lead to any more pronounced structural effect in S compared to saturated monomeric hACE2 binding. These experiments provide mechanistic insights into receptor-induced activation of Sarbecovirus spike proteins.

Detergent modulates the conformational equilibrium of SARS-CoV-2 Spike during cryo-EM structural determination.

The Spike glycoprotein of SARS-CoV-2 mediates viral entry into the host cell via the interaction between its receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2). Spike RBD has been reported to adopt two primary conformations, a closed conformation in which the binding site is shielded and unable to interact with ACE2, and an open conformation that is capable of binding ACE2. Many structural studies have probed the conformational space of the homotrimeric Spike from SARS-CoV-2. However, how sample buffer conditions used during structural determination influence the Spike conformation is currently unclear. Here, we systematically explored the impact of commonly used detergents on the conformational space of Spike. We show that in the presence of detergent, the Spike glycoprotein predominantly occupies a closed conformational state during cryo-EM structural determination. However, in the absence of detergent, such conformational compaction was neither observed by cryo-EM, nor by single-molecule FRET designed to visualize the movement of RBD in solution in real-time. Our results highlight the highly sensitive nature of the Spike conformational space to buffer composition during cryo-EM structural determination, and emphasize the importance of orthogonal biophysical approaches to validate the structural models obtained.

S:D614G and S:H655Y are gateway mutations that act epistatically to promote SARS-CoV-2 variant fitness.

SARS-CoV-2 variants bearing complex combinations of mutations that confer increased transmissibility, COVID-19 severity, and immune escape, were first detected after S:D614G had gone to fixation, and likely originated during persistent infection of immunocompromised hosts. To test the hypothesis that S:D614G facilitated emergence of such variants, S:D614G was reverted to the ancestral sequence in the context of sequential Spike sequences from an immunocompromised individual, and within each of the major SARS-CoV-2 variants of concern. In all cases, infectivity of the S:D614G revertants was severely compromised. The infectivity of atypical SARS-CoV-2 lineages that propagated in the absence of S:D614G was found to be dependent upon either S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y are only detected in SARS-CoV-2, which is also distinguished by a polybasic S1/S2 cleavage site. Genetic and biochemical experiments here showed that S:Q613H, S:D614G, and S:H655Y each stabilize Spike on virions, and that they are dispensable in the absence of S1/S2 cleavage, consistent with selection of these mutations by the S1/S2 cleavage site. CryoEM revealed that either S:D614G or S:H655Y shift the Spike receptor binding domain (RBD) towards the open conformation required for ACE2-binding and therefore on pathway for infection. Consistent with this, an smFRET reporter for RBD conformation showed that both S:D614G and S:H655Y spontaneously adopt the conformation that ACE2 induces in the parental Spike. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y are convergent adaptations to the polybasic S1/S2 cleavage site which stabilize S1 on the virion in the open RBD conformation and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations.

Diagnostic evaluation of nCoV-QS, nCoV-QM-N, and nCoV-OM detection kits based on rRT-PCR for detection of SARS-CoV-2 in Ecuador.

Background: Ecuador was harshly impacted by COVID-19, in the region was the epicenter of the pandemic with the highest mortality rates and with the lowest rates of processed samples. Real-time reverse transcription PCR assays are essential to identify and manage the SARS-CoV-2 outbreak. Because of the global emergency, in Ecuador several commercial kits were introduced for use without clinical validation. In this manner, having the need to perform an evaluation with clinical samples before use for population screening. Objective: This study aimed to evaluate the diagnostic performance of the nCoV-QS, nCoV-QM-N, nCoV-OM detection kits lately available in Ecuador, against the LightMix E/RdRp kit using nasopharyngeal swab (NPS) samples. Materials and methods: 198 nasopharyngeal samples were used (66 fresh NPS and 132 RNA stored samples). All samples were analyzed for SARS-CoV-2 with nCoV-QS, nCoV-QM-N, nCoV-OM detection kits and compared the concordance (Cohen's Kappa index, positive percentage agreement and negative percentage agreement) to LightMix E/RdRp as reference detection kit. Results: The 198 samples presented strong concordance (96% nCoV-QM-N, 100% nCoV-OM and 100% nCoV-QS). The individual performance of each gene showed that the nCoV-OM kit had a higher number of samples detected with the ORF3a (52.5%) and N (53.5%) genes. The combined genes demonstrated that ORF3a/N of nCoV-OM and nCoV-QS kits presented a higher percentage of detection with 52.5% and 48.5%, respectively. Finally, the detection rate and cycle threshold were not different between ORF3a, N, and E target genes. Conclusion: The nCoV-QS, nCoV-QM-N, and nCoV-OM Detection kits have comparable diagnostic performance to the emergency approved LightMix E/RdRp kit for SARS-CoV-2 detection in suspected COVID-19 patients.

Real-world 6-month outcomes of minimally invasive aortic valve replacement with the EDWARDS INTUITY Elite valve system.

OBJECTIVES: We report on real-world safety and performance outcomes of minimally invasive rapid-deployment aortic valve replacement using the EDWARDS INTUITY Elite aortic valve system. METHODS: The study valve system was used in a European, prospective, multicentre post-market study. Various procedural, haemodynamic and clinical outcomes were evaluated through 6 months of post-implant. RESULTS: A total of 276 patients out of 280 (98.6%) enrolments were successfully implanted with the study valve using a minimally invasive approach between February 2016 and April 2017. Of these 276 patients, 240 (87%) underwent partial sternotomy and 36 (13%) patients underwent right thoracotomy. Mean cross-clamp time was 51.9 [standard deviation (SD): 16.0] min. From baseline to 6 months, the mean effective orifice area increased from 0.8 (SD: 0.3) to 1.8 (SD: 0.6) cm2 and the mean systolic gradient decreased from 46.0 (SD: 14.1) to 8.8 (SD: 3.7) mmHg. After 6 months, 70.7% and 26.4% of patients were in New York Heart Association class I and II, respectively. Freedom from death, major bleeding, major paravalvular leak, reoperation and device explant at 6 months were 96.0%, 98.5%, 98.8%, 99.2% and 99.2%, respectively. CONCLUSIONS: These results demonstrate that the study valve is a safe and effective choice for patients undergoing aortic valve replacement via minimally invasive surgery. NAME AND REGISTRATION OF REGISTRY: MISSION (Assessing clinical outcomes using the EDWARDS INTUITY Elite Valve System in isolated AVR using Minimally InvaSive Surgery In a EurOpean multi-ceNter, active, post-market registry). clinicaltrials.gov ID #NCT02907463.

Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging, we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and in the presence of the D614G mutation. We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

Comparative analysis of neuroinvasion by Japanese encephalitis virulent and vaccine viral strains in an in vitro model of human blood-brain barrier.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.

Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts.

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.

Relationship of Serum Levels of IL-17, IL-18, TNF-α, and Lung Function Parameters in Patients with COPD, Asthma-COPD Overlap, and Bronchial Asthma.

Determination of markers of systemic inflammation is one of the important directions in the study of pathogenesis and improvement of diagnosis of chronic obstructive pulmonary disease (COPD), asthma-COPD overlap (ACO), and bronchial asthma (BA). The aim of our work was a comparative study of the features of changes in serum levels of IL-17, IL-18, and TNF-α in patients with COPD, ACO, and BA with various severity of the disease, as well as evaluation of the relationship between the level of these cytokines and lung ventilation function. A total of 147 patients with COPD (n = 58), ACO (n = 57), and BA (n = 32) during a stable period have been examined in this study. The control group included 21 healthy nonsmokers with similar sex-age indicators. Serum levels of IL-17, IL-18, and TNF-α were determined by ELISA. The concentrations of these cytokines in the circulation in the studied patients with COPD, ACO, and BA were higher than those in healthy nonsmokers (p ≤ 0.001). IL-17 and IL-18 levels in the blood serum were comparable in all examined patients. The mean TNF-α concentrations in the circulation in COPD and ACO were significantly higher than those in BA (p < 0.001). In patients with COPD, the levels of IL-17 and TNF-α increased progressively against the background of a decrease in numerous spirometric indicators, which allows us to consider these cytokines as systemic biomarkers of disease severity. In BA, the inverse correlations between the level of IL-17 and FEV1/FVC (%) and FEV1 have been found. In patients with ACO, the increase in IL-18 levels was associated with a decrease in FEV1 and TNF-α with FEV1/FVC (%). These findings indicate that IL-17, IL-18, and TNF-α can participate in the mechanisms of systemic inflammation and the genesis of disorders of airway obstruction in COPD, AСO, and BA. An increase in the levels of IL-17 and TNF-α may be associated with impaired bronchial patency in COPD and BA. The established associations of the IL-18 concentration in the blood serum and FEV1 only in patients with ACO allow using the level of IL-18 as a potential marker of the degree of impaired airway obstruction in this disease.

PPARGC1A promoter DNA-methylation level and glucose metabolism in Ecuadorian women with Turner syndrome.

OBJECTIVES: Reduced gene expression of PPARGC1A in subjects with insulin resistance (IR) has been reported. Insulin resistance occurs early on the course of Turner syndrome (TS). The main objective of this study was to evaluate the relationship between PPARGC1A promoter DNA methylation status in lymphocytes and insulin sensitivity and secretion in Ecuadorian females with TS. METHODS: We examined a cohort of 34 Ecuadorian patients with TS along with a sex-, age- and BMI-matched reference group. All subjects received a standard 75 g oral glucose tolerance test. Insulin resistance and secretion indices were calculated. The PPARGC1A methylated DNA/unmethylated DNA ratio and mitochondrial content (mtDNA/nDNA ratio) were further determined. RESULTS: Notably, the PPARGC1A DNA methylation level was significantly higher in TS subjects than the reference group and correlated with IR indices. Conversely, mitochondrial content was significantly lower in the study group than healthy controls and negatively correlated with the PPARGC1A methylated DNA/unmethylated DNA ratio in TS individuals. PPARGC1A promoter DNA methylation status contributed to 20% of the total variability in Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) independently of BMI or age in TS subjects. CONCLUSIONS: Our collective findings suggest that expression of PPARGC1A and lower mitochondrial number affect the metabolic phenotype in TS subjects.

Defining the multiplicity and time of infection for the production of Zaire Ebola virus-like particles in the insect cell-baculovirus expression system.

The Ebola virus disease is a public health challenge. To date, the only available treatments are medical support or the emergency administration of experimental drugs. The absence of licensed vaccines against Ebola virus impedes the prevention of infection. Vaccines based on recombinant virus-like particles (VLP) are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the highest mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein concentrations and improve assembly into VLP have not been determined to date. Here, we used a design of experiments (DoE) approach to determine the best MOI and TOI for three recombinant baculoviruses: bac-GP, bac-VP40 and bac-NP, each coding for one of the main structural proteins of ZEBOV. We identified two conditions where the simultaneous expression of the three recombinant proteins was observed. Interestingly, a temporal and stoichiometric interplay between the three structural proteins was observed. VP40 was required for the correct assembly of ZEBOV-VLP. High NP concentrations reduced the accumulation of GP, which has been reported to be necessary for inducing a protective immune response. Electron microscopy showed that the ZEBOV-VLP produced were morphologically similar to the native virus micrographs previously reported in the literature. A strategy for producing ZEBOV in insect cells, which consists in using a high MOI of bac-VP40 and bac-GP, and reducing expression of NP, either by delaying infection or reducing the MOI of bac-NP, was the most adequate for the production of VLP.

Most rotavirus strains require the cation-independent mannose-6-phosphate receptor, sortilin-1, and cathepsins to enter cells.

Cathepsins, endosomal acid proteases, are transported from the trans-Golgi network to late endosomes by the mannose-6-phosphate receptor (M6PR). We have previously demonstrated that some rotavirus strains, like UK, Wa, WI61, DS-1, and YM, require the cation-dependent (CD-) M6PR and cathepsins to enter from late endosomes to the cytoplasm in MA104 cells, while other strains, like the simian strain RRV, which enter cells from maturing endosomes, do not. However, the role of other trans-Golgi network-late endosome transporters, such as the cation-independent (CI-) M6PR and sortillin-1, has not been evaluated. In this work, we found that several rotavirus strains that require the CD-M6PR for cell entry are also dependent on CI-M6PR and sortilin-1. Furthermore, we showed that the infectivity of all these rotavirus strains also requires cathepsins to enter not only MA104 cells, but also human intestinal Caco-2 cells. This study identifies sortilin-1 as a novel cell factor necessary for the infectivity of a virus; in addition, our results strongly suggest that cathepsins could be common cell factors needed for the infectivity of most rotavirus strains.

Targeting antigens to Dec-205 on dendritic cells induces a higher immune response in chickens: Hemagglutinin of avian influenza virus example.

It is widely known that targeting a variety of antigens to the DEC-205 receptor on dendritic cells (DCs) significantly potentiate immunity. This communication reports the development of a new murine monoclonal antibody (mAb) against the chicken DEC-205, using as immunogen the carbohydrate recognition domain-2 (CRD-2) heterologously expressed. This mAb recognizes a protein band of 250kDa by immunoprecipitation analysis and shows strong cross-reactivity with human and pig DEC-205. Furthermore, the hemagglutinin (HA) of avian influenza H5N2 virus was cloned and expressed using insect cell-baculovirus expression system. We chemically conjugated the anti-chicken DEC-205 antibody with the highly purified HA to direct the antigen to the dendritic cells and evaluate the immune response elicited in vivo by this conjugate. A single dose of chemical conjugate was sufficient to elicit a strong immune response in chickens as early as fourteen days after priming. In addition, the conjugate induced an earlier and higher response compared to unconjugated HA. These results suggest that the strategy described here has potential to be used in the future design and development of successful vaccines against different chicken infectious diseases with direct impact in biotechnology and veterinary fields.

Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell.

UNLABELLED: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCE: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

The spike protein VP4 defines the endocytic pathway used by rotavirus to enter MA104 cells.

Rotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway.