Degradation Of Virions Research Articles

Naturally-associated bacteria modulate Orsay virus infection of Caenorhabditis elegans.

Microbes associated with an organism can significantly modulate its susceptibility to viral infections, but our understanding of the influence of individual microbes remains limited. The nematode Caenorhabditis elegans is a model organism that in nature inhabits environments rich in bacteria. Here, we examine the impact of 71 naturally associated bacteria on C. elegans susceptibility to its only known natural virus, the Orsay virus. Our findings reveal that viral infection of C. elegans is significantly influenced by monobacterial environments. Compared to an Escherichia coli environmental reference, the majority of tested bacteria reduced C. elegans susceptibility to viral infection. This reduction is not caused by virion degradation or poor animal nutrition by the bacteria. The repression of viral infection by the bacterial strains Chryseobacterium JUb44 and Sphingobacterium BIGb0172 does not require the RIG-I homolog DRH-1, which is known to activate antiviral responses such as RNA interference and transcriptional regulation. Our research highlights the necessity of considering natural biotic environments in viral infection studies and opens the way future research on host-microbe-virus interactions.

Folliculin Prevents Lysosomal Degradation of Human Papillomavirus To Support Infectious Cell Entry.

Human papillomavirus (HPV) infects epithelial basal cells in the mucosa and either proliferates with the differentiation of the basal cells or persists in them. Multiple host factors are required to support the HPV life cycle; however, the molecular mechanisms involved in cell entry are not yet fully understood. In this study, we performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout (KO) screen in HeLa cells and identified folliculin (FLCN), a GTPase-activating protein for Rag GTPases, as an important host factor for HPV infection. The introduction of single guide RNAs for the FLCN gene into HeLa, HaCaT, and ectocervical Ect1 cells reduced infection by HPV18 pseudovirions (18PsVs) and 16PsVs. FLCN KO HeLa cells also exhibited strong resistance to infection with 18PsVs and 16PsVs; nevertheless, they remained highly susceptible to infections with vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus. Immunofluorescence microscopy revealed that the numbers of virions binding to the cell surface were slightly increased in FLCN KO cells. However, virion internalization analysis showed that the internalized virions were rapidly degraded in FLCN KO cells. This degradation was blocked by treatment with the lysosome inhibitor bafilomycin A1. Furthermore, the virion degradation phenotype was also observed in Ras-related GTP-binding protein C (RagC) KO cells. These results suggest that FLCN prevents the lysosomal degradation of incoming HPV virions by enhancing lysosomal RagC activity. IMPORTANCE Cell entry by human papillomavirus (HPV) involves a cellular retrograde transport pathway from the endosome to the trans-Golgi network/Golgi apparatus. However, the mechanism by which this viral trafficking is safeguarded is poorly understood. This is the first study showing that the GTPase-activating protein folliculin (FLCN) protects incoming HPV virions from lysosomal degradation and supports infectious cell entry by activating the Rag GTPases, presumably through the suppression of excessive lysosomal biosynthesis. These findings provide new insights into the effects of small GTPase activity regulation on HPV cell entry and enhance our understanding of the HPV degradation pathway.

Lipid phosphatase SAC1 suppresses hepatitis B virus replication through promoting autophagic degradation of virions

Phosphatidylinositol lipids play vital roles in lipid signal transduction, membrane recognition, vesicle transport, and viral replication. Previous studies have revealed that SAC1-like phosphatidylinositol phosphatase (SACM1L/SAC1), which uses phosphatidylinositol-4-phosphate (PI4P) as its substrate, greatly affects the replication of certain bacteria and viruses in vitro. However, it remains unclear whether and how SAC1 modulates hepatitis B virus (HBV) replication in vitro and in vivo. In the present study, we observed that SAC1 silencing significantly increased HBV DNA replication, subviral particle (SVP) expression, and secretion of HBV virions, whereas SAC1 overexpression exerted the opposite effects. Moreover, SAC1 overexpression inhibited HBV DNA replication and SVP expression in a hydrodynamic injection-based HBV-persistent replicating mouse model. Mechanistically, SAC1 silencing increased the number of HBV-containing autophagosomes as well as PI4P levels on the autophagosome membrane. Moreover, SAC1 silencing blocked autophagosome-lysosome fusion by inhibiting the interaction between synaptosomal-associated protein 29 and vesicle-associated membrane protein 8. Collectively, our data indicate that SAC1 significantly inhibits HBV replication by promoting the autophagic degradation of HBV virions. Our findings support that SAC1-mediated phospholipid metabolism greatly modulates certain steps of the HBV life-cycle and provide a new theoretical basis for antiviral therapy.

Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms.

Human cytomegalovirus (HCMV) is the top viral cause of birth defects worldwide, and current therapies have high toxicity. We previously reported that the mTOR-independent autophagy-inducing disaccharide trehalose inhibits HCMV replication in multiple cell types. Here, we examine the mechanism of inhibition and introduce the autophagy inducer SMER28 as an additional inhibitor of HCMV acting through a different mechanism. We find that trehalose induces vacuolation and acidification of vacuoles and that debris, including debris with an appearance consistent with that of abnormal virions, is present in multivesicular bodies. Trehalose treatment increased the levels of Rab7, a protein required for lysosomal biogenesis and fusion, and slightly decreased the levels of Rab11, which is associated with recycling endosomes. We also present evidence that trehalose can promote autophagy without altering cellular glucose uptake. We show that SMER28 inhibits HCMV at the level of early protein production and interferes with viral genome replication in a cell type-dependent fashion. Finally, we show that SMER28 treatment does not cause the vacuolation, acidification, or redistribution of Rab7 associated with trehalose treatment and shows only a modest and cell type-dependent effect on autophagy. We propose a model in which the reciprocal effects on Rab7 and Rab11 induced by trehalose contribute to the redirection of enveloped virions from the plasma membrane to acidified compartments and subsequent degradation, and SMER28 treatment results in decreased expression levels of early and late proteins, reducing the number of virions produced without the widespread vacuolation characteristic of trehalose treatment.IMPORTANCE There is a need for less toxic HCMV antiviral drugs, and modulation of autophagy to control viral infection is a new strategy that takes advantage of virus dependence on autophagy inhibition. The present study extends our previous work on trehalose by showing a possible mechanism of action and introduces another autophagy-inducing compound, SMER28, that is effective against HCMV in several cell types. The mechanism by which trehalose induces autophagy is currently unknown, although our data show that trehalose does not inhibit cellular glucose uptake in cells relevant for HCMV replication but instead alters virion degradation by promoting acidic vacuolization. The comparison of our cell types and those used by others highlights the cell type-dependent nature of studying autophagy.

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

Vpr Promotes Macrophage-Dependent HIV-1 Infection of CD4+ T Lymphocytes.

Vpr is a conserved primate lentiviral protein that promotes infection of T lymphocytes in vivo by an unknown mechanism. Here we demonstrate that Vpr and its cellular co-factor, DCAF1, are necessary for efficient cell-to-cell spread of HIV-1 from macrophages to CD4+ T lymphocytes when there is inadequate cell-free virus to support direct T lymphocyte infection. Remarkably, Vpr functioned to counteract a macrophage-specific intrinsic antiviral pathway that targeted Env-containing virions to LAMP1+ lysosomal compartments. This restriction of Env also impaired virological synapses formed through interactions between HIV-1 Env on infected macrophages and CD4 on T lymphocytes. Treatment of infected macrophages with exogenous interferon-alpha induced virion degradation and blocked synapse formation, overcoming the effects of Vpr. These results provide a mechanism that helps explain the in vivo requirement for Vpr and suggests that a macrophage-dependent stage of HIV-1 infection drives the evolutionary conservation of Vpr.

Dysregulation of Autophagy in Murine Fibroblasts Resistant to HSV-1 Infection

The mouse L cell mutant, gro29, was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1). gro29 cells are fully susceptible to HSV-1 infection, however, they produce 2000-fold less infectious virus than parental L cells despite their capacity to synthesize late viral gene products and assemble virions. Because productive HSV-1 infection is presumed to result in the death of the host cell, we questioned how gro29 cells might survive infection. Using time-lapse video microscopy, we demonstrated that a fraction of infected gro29 cells survived infection and divided. Electron microscopy of infected gro29 cells, revealed large membranous vesicles that contained virions as well as cytoplasmic constituents. These structures were reminiscent of autophagosomes. Autophagy is an ancient cellular process that, under nutrient deprivation conditions, results in the degradation and catabolism of cytoplasmic components and organelles. We hypothesized that enhanced autophagy, and resultant degradation of virions, might explain the ability of gro29 to survive HSV-1 infection. Here we demonstrate that gro29 cells have enhanced basal autophagy as compared to parental L cells. Moreover, treatment of gro29 cells with 3-methyladenine, an inhibitor of autophagy, failed to prevent the formation of autophagosome-like organelles in gro29 cells indicating that autophagy was dysregulated in these cells. Additionally, we observed robust co-localization of the virion structural component, VP26, with the autophagosomal marker, GFP-LC3, in infected gro29 cells that was not seen in infected parental L cells. Collectively, these data support a model whereby gro29 cells prevent the release of infectious virus by directing intracellular virions to an autophagosome-like compartment. Importantly, induction of autophagy in parental L cells did not prevent HSV-1 production, indicating that the relationship between autophagy, virus replication, and survival of HSV-1 infection by gro29 cells is complex.

Unique Characteristics of AAV1, 2, and 5 Viral Entry, Intracellular Trafficking, and Nuclear Import Define Transduction Efficiency in HeLa Cells

Biological differences between recombinant adeno-associated virus (rAAV) serotypes define their efficiencies in expressing a transgene in a particular target cell. Few studies have directly compared how differences in viral entry, intracellular trafficking, and nuclear import of rAAV serotypes influence the effectiveness of transduction in the same cell type. We evaluated these characteristics for three rAAV serotypes in HeLa cells, using biochemical techniques and fluorescence-based detection of multiple serotypes in the same cell. Although rAAV2 exhibited the slowest entry, intracellular trafficking, and nuclear import among the three serotypes, it elicited the highest levels of transduction. Conversely, rAAV1 exhibited more rapid entry and nuclear import than the other serotypes, yet was ineffective at transducing HeLa cells due to impaired capsid disassembly in the nucleus. rAAV5, which entered the cell less rapidly than rAAV1, was imported efficiently into the nucleus, but then rapidly degraded, resulting in poor transduction of HeLa cells. We conclude that rAAV1, 2, and 5 utilize distinct mechanisms for intracellular trafficking, and that post-nuclear events play an important role in determining the efficiency of HeLa cell transduction by these serotypes. Thus, overcoming post-nuclear barriers that limit uncoating and/or promote virion degradation may enhance the efficiency of certain AAV serotypes.

Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21).

Antibodies provide effective antiviral immunity despite the fact that viruses escape into cells when they infect. Here we show that antibodies remain attached to viruses after cell infection and mediate an intracellular immune response that disables virions in the cytosol. We have discovered that cells possess a cytosolic IgG receptor, tripartite motif-containing 21 (TRIM21), which binds to antibodies with a higher affinity than any other IgG receptor in the human body. TRIM21 rapidly recruits to incoming antibody-bound virus and targets it to the proteasome via its E3 ubiquitin ligase activity. Proteasomal targeting leads to rapid degradation of virions in the cytosol before translation of virally encoded genes. Infection experiments demonstrate that at physiological antibody concentrations TRIM21 neutralizes viral infection. These results reveal an intracellular arm of adaptive immunity in which the protection mediated by antibodies does not end at the cell membrane but continues inside the cell to provide a last line of defense against infection.

Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV‐1

The outcome following HIV infection depends on the nature and durability of the HIV-specific T cell response induced initially. The activation of protective T cell responses depends upon dendritic cells (DC), antigen-presenting cells which have the capacity to process and present viral antigens. DC pulsed with aldrithiol-2-inactivated HIV and delivered in vivo were reported to induce immune responses and promote virologic control in chronically HIV-1-infected subjects. To gain an understanding of this phenomenon, we characterized the steps involved in the presentation of antigens derived from aldrithiol-2-treated vs. infectious HIV-1 by DC. Antigen presentation, on both MHC class I and II, was independent of DC-specific ICAM-3-grabbing integrin, DEC-205 and macrophage mannose receptor, C-type lectins expressed by the DC. Inhibitor studies showed that presentation on MHC class I was dependent on viral fusion in a CD4/coreceptor-dependent manner, both at the cell surface and within endosomes, and access to the classical endosomal processing pathway. MHC class II presentation of HIV-associated antigens was dependent on active endocytosis, probably receptor-mediated, and subsequent degradation of virions in acidified endosomes in the DC. Our study brings forth new facts regarding the binding, uptake, and processing of chemically inactivated virions leading to efficient antigen presentation and should aid in the design of more effective HIV vaccines.

Adeno-associated virus (AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation

Transduction of the vascular endothelium by adeno-associated virus (AAV) vectors would have broad appeal for gene therapy. However, levels of transduction by AAV serotype-2 are low, an observation linked to deficiencies in endothelial cell binding, sequestration of virions in the extracellular matrix and/or virion degradation by the proteasome. Strategies to improve transduction of endothelial cells include AAV-2 capsid targeting using small peptides isolated by phage display or the use of alternate serotypes. Previously, we have shown that AAV serotypes-3 through -6 transduce endothelial cells with poor efficiency. Recently, AAV serotypes-7 and -8 have been shown to mediate efficient transduction of the skeletal muscle and liver, respectively, although their infectivity profile for vascular cells has not been addressed. Here, we show that AAV-7 and -8 also transduce endothelial cells with poor efficiency and the levels of transgene expression are markedly enhanced by inhibition of the proteasome. In both cases proteasome blockade enhances the nuclear translocation of virions. We further show that this is vascular cell-type selective since transduction of smooth muscle cells is not sensitive to proteasome inhibition. Analysis in intact blood vessels corroborated these findings and suggests that proteasome degradation is a common limiting factor for endothelial cell transduction by AAV vectors.

Biophysical Studies on the RNA Cores of Satellite Tobacco Mosaic Virus

Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90°C. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2–12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.

Inactivation of the yellow strain of cucumber mosaic virus by pancreatic ribonuclease.

The infectivity of cucumber mosaic virus Y strain (CMV-Y) decreased after mixing with bovine pancreatic ribonuclease (RNase). The amount of infectivity lost was dependent on the virus to enzyme ratio in the mixture and on the pH used for incubation. The enzyme seemed to bind at random to the surface of virus particles. Binding was partially reversed by pH adjustment. Most of the RNase which attached to the virus at pH 6 appeared to be released at pH 8. As the proportion of RNase in mixtures increased, the virus particles and enzyme formed complex aggregates. Degradation of virions occurred only at very high RNase concentrations. Degradation occurred more rapidly at pH 6 than pH 8. The infectivity loss upon addition of small amounts of RNase appeared not to be caused by aggregation or virion degradation. It is suggested that RNase attached to the virus prevents some processes necessary for initiation of infection or attacks the uncoated RNA.

Ultrastructure and infectivity of tissues from normal and immunodepressed chickens inoculated with turkey herpesvirus

A study was made of the ultrastructural changes and infectivity in tissues of normal and cyclophosphamide (Cy)-treated chickens inoculated with turkey herpesvirus (HVT). In untreated infected chickens, the ultrastructural changes in lung and lymphoid tissues were characterized mainly by invaginations in the nuclear and plasma membranes and an increased number of lysosomes in the cytoplasm, and they were observed in only a small proportion of cells. A few naked herpes virions were seen in the nucleus and cytoplasm and the virus particles in the cytoplasm often appeared degenerated. Enveloped virus was not detected in any tissue. In Cy-treated chickens, the virion degradation was rarely seen. Increased lysosomal activity in the phagocytic cells of untreated chickens was believed to account for the cytoplasmic degradation of the naked virions. In untreated chickens, the cell-associated infectivity was first detected in buffy coat, lung, spleen and thymus 2 days after inoculation. No virus was detected, either by cell culture or by electron microscopy, in skin or feather quill-tips of infected chickens up to 3 weeks after inoculation. The virus titres in tissues of Cy-treated chickens were considerably higher than those in the corresponding control on the 10th day post-inoculation.

Structural polypeptides of Orthopoxvirus: their distribution in various members and location within the virion.

The structural polypeptides of accepted species and recently isolated members of the genus Orthopoxvirus have been examined by SDS-polyacrylamide gel electrophoresis. The viruses shared many polypeptides but some differences were found. The viruses could be divided into a vaccinia group (including buffalopox, 'Lenny' and MK-10), an ectromelia group (including elephant virus and Moscow virus), cowpox, camelpox and monkeypox. Minor differences were found in the polypeptides of monkeypox virus strains from human and monkey outbreaks. Controlled degradation of virions showed that the polypeptides which enabled the viruses to be differentiated were located in the surface and sub-surface layers. The cores of the viruses all gave the same complex polypeptide pattern.

Synthesis and distribution of vesicular stomatitis virus-specific polypeptides in the absence of progeny production

In cultures of Chinese hamster ovary cells infected by vesicular stomatitis virus (VSV), viral polypeptides can be found in cellular, virion-associated, and soluble fractions. In order to determine whether or not the soluble polypeptides resulted from degradation of virions, progeny formation was inhibited and the distribution of polypeptides in these fractions was examined. Synthesis of infectious progeny was prevented in three ways: (1) addition of defective interfering (DI) particles; (2) utilization of a replication-minus mutant, tsG41; and (3) utilization of a glycoprotein mutant, ts045. In experiments involving either the coinfection of standard infectious particles and DI particles or the infection of cells at the nonpermissive condition for tsG41, overall virus-specific RNA synthesis was reduced by 80%, whereas the appearance of extracellular soluble viral polypeptides was not greatly inhibited. Cell-associated polypeptides were also present in normal amounts. By using tsO45, it was shown that there was no virion maturation but, surprisingly, the appearance of soluble viral polypeptides was enhanced. Therefore, the distribution of viral proteins in the cell-associated and extracellular soluble fractions was not dependent on the production of infectious progeny. The major soluble viral polypeptide was a glycoprotein which migrated more rapidly than the virion-associated glycoprotein; these two glycoproteins had about the same amount of carbohydrate, even when the soluble glycoprotein was synthesized by tsO45 under nonpermissive conditions. These results lead to the hypothesis that G protein interaction with the plasma membrane exists in three different states: (1) an initial very unstable association; (2) possible stabilization at the cell surface via interactions with another membrane protein called matrix protein; and (3) aggregation of G protein at the cell surface initiated by nucleocapsids interacting through the matrix proteins. The first two stages of this interaction probably result in the shedding of G protein from the cell surface.

Evidence for the presence of RNA in the purified virions of vaccinia virus.

Vaccinia virus, strain WR, was propagated in HeLa cells, L mouse fibroblats, or primary chicken embryo fibroblasts in the presence of [5- (3)H]uridine. Carefully purified virions were found to contain significant amounts of labeled trichloroacetic acid-precipitable material which was rendered acid soluble when digested with pancreatic RNase or hydrolyzed in alkali. Controlled degradation of virions with Nonidet P-40 and 2-mercaptoethanol demonstrated that 65 to 80% of the [5- (3)H]uridine-labeled molecules resided in the viral core. When the total nucleic acids were extracted from viral cores prepared from virions propagated in HeLa cells, 30 to 50% of the total incorporated [5- (3)H]uridine was found in RNA; in L mouse fibroblasts, 40 to 50%; in primary chicken embryo fibroblasts, 50 to 60%. The RNA molecules do not appear to be covalently linked to the viral DNA genome but sediment in sodium dodecyl sulfate-sucrose gradients as 8 to 10S species relative to ribosomal RNA.

Structural proteins of adenoviruses. X. Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2.

Abstract With high resolution SDS-polyacrylamide gel electrophoresis ( Maizel, 1971 ) and a new method to extract the basic proteins, it was ascertained that adenovirus type 2 contains at least 10 distinct polypeptides (II, III, IIIa, IV–X) and possibly more. Five proteins (V, VI, VII, VIII, and X) were purified by selective extraction in urea at high ionic strength, and low pH followed by preparative polyacrylamide electrophoresis toward the cathode at pH 3.4. Antisera, produced against proteins V, VI, and VII were used to reveal that these proteins were antigenically distinct and unrelated to hexons, pentons, and fibers. The location of the polypeptides was investigated by two methods of virion degradation ( Prage et al. , 1970 ). Polypeptides V and VII were associated with the DNA-containing core. Polypeptide VI appeared to be associated with all hexons of the virion. Hexons from the triangular facets of the capsid contained in addition to polypeptides II and VI, polypeptide IX, and possibly also polypeptide VIII. Hexons purified from infected cells contained only polypeptide II. Polypeptide IIIa was preferentially released together with the peripentonal hexons.

Gel electrophoresis of reverse transcriptase activity of murine mammary tumour virions.

Milk-borne murine mammary tumour virus induces mammary tumours in mice1 and possesses 60–70S RNA2 and RNA-dependent DNA polymerase activity3 characteristic of oncornaviruses4. The discovery of similar particles in some human milks5 and their possible correlation to reverse transcriptase activity6 has recently become less certain, possibly due to the small sample size examined or to the degradation of virions isolated from some human milks7.

Dengue Virions and Antigens in Brain and Serum of Infected Mice

The temporal relationships of the production of infectious dengue-2 virus and its antigens were investigated in intracerebrally infected suckling mice. Infectious virus, a slowly sedimenting noninfectious hemagglutinin (SHA), and a noninfectious soluble complement-fixing antigen (SCF) were found in the brain. Serum contained high concentrations of SCF antigen relative to infectivity when compared to SCF to infectivity ratios in the brain. Degradation of virions by Tween-80 and ether produced two antigens with sedimentation characteristics similar to the noninfectious antigens occurring naturally in infected tissues. However, the virionderived SHA differed from native SHA when examined by electron microscopy and by equilibrium centrifugation in cesium chloride. Virion-derived SCF (as well as the virions and both SHA antigens) was denatured by sodium lauryl sulfate (SLS) and 2-mercaptoethanol (2-ME), whereas native SCF retained its complement-fixing activity. SLS and 2-ME treatment of dengue-1 and dengue-2 sucrose-acetone antigens increased their serotypic specificity. The hemagglutinin present in sucrose-acetone antigens was predominantly native SHA.