Glucose-regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers, and neurological disorders.

The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.

Potential interactions of SARS-CoV-2 with human cell receptors in the skin: Understanding the enigma for a lower frequency of skin lesions compared to other tissues.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a new public health problem, with a total of 10.577.263 documented COVID-19 cases worldwide and 513.441 deaths up to the present date. Few cases of disease-related cutaneous manifestations have been reported in the literature, and such manifestations are scarce. Integumentary manifestations from COVID-19 include exanthemas and papular dermatoses, urticarial eruptions, atopic dermatitis, vesiculobullous lesions and skin signs of hypercoagulable states, such as acral ischaemia, livedo and retiform purpura. Most common extracutaneous manifestations from the disease include headache, cough, anosmia, ageusia, fever, dyspnoea, nausea, diarrhoea and cardiovascular events. The objectives of this review were to discuss the role of human cell receptors described as interaction targets of SARS-CoV-2, as well to understand the current state of knowledge on skin expression of these receptors, in order to substantiate future research. The authors present a thorough literature review on SARS-CoV-2 and its possible interaction with cell receptors and human tissues including the skin. They discuss a molecular hypothesis to explain the lower prevalence of dermatological manifestations from direct SARS-CoV-2 infection. Distinct human cell receptors binding the virus appear to be less expressed in the skin compared to other organs. Additionally, the presence of resolvins and the disintegrin metalloprotease ADAM17 provide a putative protection to the skin, explaining the majority of COVID-19 manifestations to be extracutaneous. This review represents an excellent opportunity for future studies using skin biopsies from COVID-19 patients to investigate molecular expression in the pathophysiology of cutaneous manifestations of the disease.

What would Sérgio Ferreira say to your physician in this war against COVID-19: How about kallikrein/kinin system?

Coronavirus disease 2019 (COVID-19) is an infectious disease with fast spreading all over the world caused by the SARS-CoV-2 virus which can culminate in a severe acute respiratory syndrome by the injury caused in the lungs. However, other organs can be also damaged. SARS-CoV-2 enter into the host cells using the angiotensin-converting enzyme 2 (ACE2) as receptor, like its ancestor SARS-CoV. ACE2 is then downregulated in lung tissues with augmented serum levels of ACE2 in SARS-CoV-2 patients. Interestingly, ACE2+ organs reveal the symptomatic repercussions, which are signals of the infection such as dry cough, shortness of breath, heart failure, liver and kidney damage, anosmia or hyposmia, and diarrhea. ACE2 exerts a chief role in the renin-angiotensin system (RAS) by converting angiotensin II to angiotensin-(1-7) that activates Mas receptor, inhibits ACE1, and modulates bradykinin (BK) receptor sensitivity, especially the BK type 2 receptor (BKB2R). ACE2 also hydrolizes des-Arg9-bradykinin (DABK), an active BK metabolite, agonist at BK type 1 receptors (BKB1R), which is upregulated by inflammation. In this opinion article, we conjecture a dialogue by the figure of Sérgio Ferreira which brought together basic science of classical pharmacology and clinical repercussions in COVID-19, then we propose that in the course of SARS-CoV-2 infection: i) downregulation of ACE2 impairs the angiotensin II and DABK inactivation; ii) BK and its metabolite DABK seems to be in elevated levels in tissues by interferences in kallikrein/kinin system; iii) BK1 receptor contributes to the outbreak and maintenance of the inflammatory response; iv) kallikrein/kinin system crosstalks to RAS and coagulation system, linking inflammation to thrombosis and organ injury. We hypothesize that targeting the kallikrein/kinin system and BKB1R pathway may be beneficial in SARS-CoV-2 infection, especially on early stages. This route of inference should be experimentally verified by SARS-CoV-2 infected mice.

[COVID-19 and its relationship with hypertension and cardiovascular disease]. / COVID-19, hipertensión y enfermedad cardiovascular.

The association between hypertension, diabetes, cardio and cerebrovascular disease and severe and fatal COVID-19, described in different countries, is remarkable. Myocardial damage and myocardial dysfunction are postulated as a possible causal nexus. Frequent findings of elevated troponin levels and electrocardiographic anomalies support this concept. On the other hand, hypotheses in favour and against a deleterious effect of angiotensin converting enzyme inhibitors and angiotensin receptor blockers, a usual treatment for cardiovascular disease, have been raised. There is currently no solid evidence and thus properly designed studies on this subject are urgently needed. In this context, patients with cardiovascular disease should especially avoid being exposed to the virus, should not self-medicate and rapidly seek medical advice should they show symptoms of infection.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses.

BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection.

BACKGROUND: The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. OBJECTIVES: We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. MATERIALS AND METHODS: Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. RESULTS: The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). CONCLUSIONS: HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread.

A polymorphism in TIM1 is associated with susceptibility to severe hepatitis A virus infection in humans.

During infection with the hepatitis A virus (HAV), most patients develop mild or asymptomatic disease. However, a small number of patients develop serious, life-threatening hepatitis. We investigated this variability in disease severity by examining 30 Argentinean patients with HAV-induced acute liver failure in a case-control, cross-sectional, observational study. We found that HAV-induced severe liver disease was associated with a 6-amino-acid insertion in TIM1/HAVCR1 (157insMTTTVP), the gene encoding the HAV receptor. This polymorphism was previously shown to be associated with protection against asthma and allergic diseases and with HIV progression. In binding assays, the TIM-1 protein containing the 157insMTTTVP insertion polymorphism bound HAV more efficiently. When expressed by human natural killer T (NKT) cells, this long form resulted in greater NKT cell cytolytic activity against HAV-infected liver cells, compared with the shorter TIM-1 protein without the polymorphism. To our knowledge, the 157insMTTTVP polymorphism in TIM1 is the first genetic susceptibility factor shown to predispose to HAV-induced acute liver failure. Furthermore, these results suggest that HAV infection has driven the natural selection of shorter forms of the TIM-1 protein, which binds HAV less efficiently, thereby protecting against severe HAV-induced disease, but which may predispose toward inflammation associated with asthma and allergy.

[Hantavirus as important emerging agents in South America]. / Hantavirus como agentes emergentes de importancia en Suramérica.

The dawning of the 20th century was marked by the emergence of new infectious disease agents and the appearance of others previously thought controlled. Both phenomena were possibly connected with ecological disturbances that led to the recognition of a dramatic climate change, of which the effects are only now becoming noticeable. Among the variety of agents to be considered, the many new viruses stand out, not only for their numerical proliferation, but also for their genetic versatility. It is this quality that provides them dexterity for evolving new strategies and adaptations to changing environmental conditions. Recently, some of the most ubiquitous and well-publicized viral agents in the American continents have been the rodent-borne viruses, and among these are the hantaviruses, etiological agents of pulmonary syndromes. Approximately 18 hantaviruses (belonging to the family Bunyaviridae), have been discovered in South America during the last 20 years, and although most of them cause persistent infections and subclinical infections in wild rodents (particularly members of the subfamily Sigmodontinae) and humans respectively; some others might also be highly lethal for humans. The goal herein is to review the state of the art regarding general aspects of hantaviruses and the diseases they cause around the world, highlighting the most recent findings in Colombia. Finally, the many unanswered questions will be recognized and highlighted concerning clinical importance and socio-economic impact of these agents on quality of public health in Colombia.

Development of a lentiviral vector system to study the role of the Andes virus glycoproteins.

To infect target cells, enveloped viruses use their virion surface proteins to direct cell attachment and subsequent entry via virus-cell membrane fusion. How hantaviruses enter cells has been largely unexplored. To study early steps of Andes virus (ANDV) cell infection, a lentiviral vector system was developed based on a Simian immunodeficiency virus (SIV) vector pseudotyped with the ANDV-Gn/Gc envelope glycoproteins. The incorporation of Gn and Gc onto SIV-derived vector particles was assessed using newly generated monoclonal antibodies against ANDV glycoproteins. In addition, sera of ANDV infected humans were able to block cell entry of the SIV vector pseudotyped with ANDV glycoproteins, suggesting that their antigenic conformation is similar to that in the native virus. The use of such SIV vector pseudotyped with ANDV-Gn/Gc glycoproteins should facilitate studies on ANDV cell entry. Along this line, it was found that depletion of cholesterol from target cells strongly diminished cell infection, indicating a possible role of lipid rafts in ANDV cell entry. The Gn/Gc pseudotyped SIV vector has several advantages, notably high titer vector production and easy quantification of cell infection by monitoring GFP reporter gene expression by flow cytometry. Such pseudotyped SIV vectors can be used to identify functional domains in the Gn/Gc glycoproteins and to screen for potential hantavirus cell entry inhibitors.

Rotaviruses require basolateral molecules for efficient infection of polarized MDCKII cells.

In this work we evaluated the ability of rotavirus strains with different receptor requirements to infect the apical and basolateral surfaces of polarized MDCKII cells. We used neuraminidase (NA)-sensitive (RRV and TFR-1) and neuraminidase-resistant (Wa and UK) viruses that differ in their use of integrins. Regardless of their receptor requirements, all virus strains tested were found to efficiently infect cells from both membrane surface domains, with preference for the basolateral domain, since: (i) disruption of tight junctions of polarized cell monolayers by calcium chelation led to a reversible increase of rotavirus infectivity, (ii) the viruses infected preferentially the cells located at the borders of microcolonies of polarized cells, and (iii) in cells grown on a permeable support all four virus strains were able to start the infection by either plasma membrane domain. Preferential infection (5-11-fold more efficiently) of the basolateral surface correlated with the neuraminidase resistance of the virus strains, but not with their requirement for integrins, which in MDCKII cells seem to be used by all four viruses. The infection of both cell surface domains by RRV was found to depend on the presence of terminal sialic acids, since its infectivity was reduced by neuraminidase treatment of the cells and it was also blocked by incubation of the virus with glycophorin A. The efficient infection through the basolateral membrane surface of polarized cells might be relevant for the pathogenesis of rotavirus, especially given the recent reports of antigenemia and extraintestinal spread of the virus in children and animal models.

Dissecting the role of integrin subunits alpha 2 and beta 3 in rotavirus cell entry by RNA silencing.

Several cell surface molecules have been implicated in rotavirus cell entry, however, their individual relevance during this process is unknown. In this work, the expression of integrins alpha2, beta2, and alpha v beta 3, the heat shock cognate protein 70, and of ganglioside GM1 in different cell lines of human and simian origin was correlated with the infectivity of four rotavirus strains. We observed that different combinations of receptor expression correlated with the infectivity of rotavirus strains, suggesting that the participation of several receptors is important for rotavirus infection. To characterize the relevance of integrins alpha2 and alpha v beta 3 in more detail, their expression was silenced using RNA interference. About 80% decrease in the cell content of integrins resulted in 15-30% decrease of infectivity of strains RRV and Wa when measured by a focus-forming assay, while there was no decrease of infectivity when measured by flow cytometry in integrin-deficient cells. Altogether these data suggest that integrins alpha2 and alpha v beta 3 do not play a major role in the rotavirus entry process.

Cellular receptors for foot and mouth disease virus.

Foot-and-mouth disease virus (FMDV), the prototype member of the Aphthovirus genus, is a single-stranded, positive-sense RNA genome virus, which affects many domestic livestock cloven-hoofed animals, causing substantial lost of milk in dairy cattle, reduction in the growth rate of meat animals, among others. It has been shown that the virus can enter to the cells using different pathways; the main one binding integrins via the clathrin-mediated endocytosis pathway, trafficking throughout the acidified endocytic vesicles, where its capsid rapidly dissociates, resulting in the release of the RNA genome, and the second one using heparan sulfate in which FMDV enters to the cells using the caveola-mediated endocytosis pathway and that caveolae can associate and traffic with endosomes. Different integrins had been involved as FMDV receptors (alphavbeta1, alphavbeta3, alpha5beta1, alphavbeta6, alphavbeta8); this review will try to resume the basic information about FMDV receptors from the last years to the present and will resume the most important in vitro and in vivo studies to elucidate the role of this receptor on the infection.

Analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, FhuA-dependent phages.

A group of previously isolated heterogeneous mEp lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. Interestingly, the FhuA host receptor was required by the majority of mEp phages (37 out of 43; approximately 85%). The cor gene, which has been reported to be involved in FhuA-dependent exclusion of lambdoid phages, was also found in most of the FhuA-dependent phages. Accordingly, no cor amplification by PCR was obtained among the six FhuA-independent mEp lambdoid phages. In contrast, it was found that around 25% of the population (10 out of 43 phages) required the specific and essential lambda N antitermination function, and the lambda site-specific DNA recombination function was observed only in two members (4.6%). Thus, a larger proportion of phages require the FhuA receptor for infection, and this is frequently correlated with the cor gene.

Evidence that the 45-kD glycoprotein, part of a putative dengue virus receptor complex in the mosquito cell line C6/36, is a heat-shock related protein.

Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.

Early steps in rotavirus cell entry.

Rotaviruses, the leading cause of severe dehydrating diarrhea in infants and young children worldwide, are non-enveloped viruses formed by three concentric layers of protein that enclose a genome of double-stranded RNA. These viruses have a specific cell tropism in vivo, infecting primarily the mature enterocytes of the villi of the small intestine. It has been found that rotavirus cell entry is a complex multistep process, in which different domains of the rotavirus surface proteins interact sequentially with different cell surface molecules, which act as attachment and entry receptors. These recently described molecules include integrins (alpha2beta1, alphavbeta3, and alphaxbeta2) and a heat shock protein (hsc70), and have been found to be associated with cell membrane lipid microdomains. The requirement for several cell molecules, which might need to be present and organized in a precise fashion, could explain the cell and tissue tropism of these viruses. This review focuses on recent data describing the interactions between the virus and its receptors, the role of lipid microdomains in rotavirus infection, and the possible mechanism of rotavirus cell entry.

Multistep entry of rotavirus into cells: a Versaillesque dance.

Rotavirus entry into a cell is a complex multistep process in which different domains of the rotavirus surface proteins interact with different cell surface molecules, which act as attachment and entry receptors. These recently described molecules include several integrins and a heat shock protein, which have been found to be associated with cell membrane lipid microdomains. The requirement during viral entry for several cell molecules, which might be required to be present and organized in a precise fashion, could explain the selective cell and tissue tropism of these viruses. This review focuses on recent data describing the virus-receptor interactions, the role of lipid microdomains in rotavirus infection and the mechanism of rotavirus cell entry.

[HIV glycoprotein 120: possible etiological agent of AIDS-associated dementia]. / La glicoproteína 120 del VIH: potencial agente etiológico de la demencia asociada al SIDA.

The AIDS or HIV associated dementia is a cognitive-motor disease, characterized by a strong deficit of several cognitive processes such as attention, memory, sensory perception, motor control among others. The HIV associated dementia affects 30% of adult to 50% of infant HIV positive subjects. Since neurons are not infected by HIV, its principal target in the brain is microglia. The pathophysiology of this syndrome, therefore, remains to be disclosed. Several hypothesis have been proposed, one of them suggests that opportunistic infections can affect the brain. Another hypothesis suggests that microglia secretes toxic products as a result of HIV infection and those are the ones causing the damage and finally, the hypothesis, suggesting that the brain is damaged as a result of the insult caused by HIV-derived proteins. In vitro studies suggest that the HIVgp120, a viral surface protein, is highly neurotoxic. For example HIVgp120 increases cytoplasmic Ca+2 by two ways: facilitating glutamate neurotransmission increasing Ca+2 conductance, and activating the IP3 pathway, facilitating Ca+2 release from the smooth endoplasmic reticulum. This Ca+2 in turn, activates several internal signaling pathways such as the MAPK pathway. We use an animal model to test the HIVgp120 effect on neurophysiological signals and behavior as well as several pharmacological approaches to prevent the HIVgp120 neurotoxic effects. This review updates with the most recent literature discussing the potential mechanisms implicated in the pathophysiology of the AIDS dementia complex. We, in addition, hope the reader will be able to correlate the clinical symptoms observed in the HIV infected subjects and the HIVgp120-induced behavioral changes observed in animal models. Likewise, we discuss the new drugs we are testing, in order to offer a new pharmacological treatment to the patient.

Biochemical characterization of rotavirus receptors in MA104 cells.

We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesis d, l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with beta-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-beta-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.

Effects of reducing, oxidizing and alkylating agents on early steps of Junin virus multiplication.

The action of reducing, oxidizing and thiol-alkylating agents on early steps of Junin virus (JV) multiplication in Vero cells was investigated. The presence of reducing agents during virus adsorption as well as incubation of viral particles with these compounds before infection enhanced JV infectivity. On the contrary, the thiol-alkylating agent 5,5' dithiobis (2-nitrobenzoic acid) and the oxidizing compound potassium periodate showed an inhibitory effect, suggesting that sulfhydryl groups, and certain sugar moieties of viral glycoproteins play an important role in the first steps of JV infection. Also enzymatic treatment of cell monolayers and addition of concanavalin A to cultures prior to infection suggest that cellular glycoproteins are involved in virus attachment.

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.