Showing papers in "Journal of Virology in 2002"

Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

Abstract: Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.

Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

Abstract: Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

Cross-Packaging of a Single Adeno-Associated Virus (AAV) Type 2 Vector Genome into Multiple AAV Serotypes Enables Transduction with Broad Specificity

Abstract: The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.

The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α1→2 Mannose Residues on the Outer Face of gp120

Abstract: The humoral immune response to infection by human immunodeficiency virus type 1 (HIV-1) is typically characterized by relatively low levels of neutralizing antibodies, particularly those with broad activity against many different isolates of the virus (6, 23, 35, 36, 46). As might perhaps be anticipated from this observation, the induction of such antibodies by vaccination has proven largely elusive (9). At the same time, interest in inducing broadly neutralizing antibodies has increased as it becomes clear that antibodies can provide considerable benefit against HIV or simian immunodeficiency virus (SIV) challenge in animal models (1, 17, 30-32, 44, 55). Fortunately, natural infection is not completely barren of lessons for vaccine design since, although HIV elicits weak cross-neutralizing responses, a small number of human monoclonal antibodies (MAbs) with broad activities have been isolated from infected individuals (7, 8, 12, 59, 60). One rational contribution to eliciting neutralizing antibodies by vaccination is then to explore the interaction of these antibodies with virus envelope at the molecular level and incorporate the information obtained into immunogen design. Here, we seek to understand the interaction of one broadly neutralizing antibody with HIV-1 envelope. For some time, only three broadly neutralizing MAbs to HIV-1 were known (5, 12). Two of these MAbs bind to the surface glycoprotein, gp120, which is the viral receptor for CD4 and chemokine receptors CCR5 and CXCR4. These MAbs are b12, which recognizes an epitope overlapping the CD4 receptor site (7, 51), and 2G12, which recognizes an epitope based around the C4/V4 region of gp120 and is highly sensitive to the presence of N-linked glycans in this region (24, 60). One MAb, 2F5, binds to an epitope involving a linear motif (ELDKWA) on the membrane proximal region of the transmembrane envelope protein gp41 (8, 24, 43, 71). Recently, two MAbs, Z13 and 4E10, have been described which recognize a region close to the C terminus of the 2F5 epitope (56, 71). Another Fab with broad neutralizing ability, X5, recognizes a region close to the coreceptor binding site on gp120 and overlapping the epitope recognized by CD4-induced MAbs, such as 17b (37a). We focus here on the MAb 2G12. In vitro, this MAb has been shown to neutralize a wide spectrum of different HIV-1 isolates (59, 60), including those from different clades, with the notable exception of clade E. In vivo, the MAb protects macaques against vaginal challenge with the chimeric virus SHIV 89.6P (32). The antibody recognizes a unique epitope in that it does not compete with any of the large panel of MAbs to gp120 that have been produced (37). The binding of 2G12 to gp120 is inhibited by a number of mutations that disrupt sequences encoding attachment of N-linked carbohydrate chains (60). These sequences are located in the C2 and C3 regions around the base of the V3 loop, the C4 region, and the V4 loop. The crystal structure of the core of gp120 suggests that the carbohydrate attachment sites are clustered together on a part of the gp120 molecule known as the “silent face” (27, 28, 66, 67). This extensive solvent-accessible face is largely covered by carbohydrate and expected to be relatively weakly immunogenic and, hence, is described as immunologically silent. Carbohydrate-rich regions of glycoproteins are generally poorly immunogenic for a number of reasons. First, carbohydrates exhibit microheterogeneity; therefore, a single protein sequence would be expected to display multiple glycoforms, leading to the dilution of any single antigenic response (52). Second, large, potentially dynamic glycans (62, 64) can cover potential protein epitopes. Third, in the case of viruses, which depend on the host glycosylation machinery since they have none of their own, the oligosaccharides attached to potential antigens are the same as those attached to host glycoproteins. Therefore, in general, the host will display tolerance towards these sugars. The difficulty in eliciting antibodies to a carbohydrate face is consistent with the apparently unique nature of MAb 2G12. Despite the probable placement of the 2G12 epitope on the silent face (or at the junction of silent and neutralizing faces) (26, 27, 49, 60), we understand relatively little about the molecular nature of the epitope. We do not know whether the epitope is exclusively carbohydrate, exclusively protein with a requirement for carbohydrate to maintain local protein structure, or some combination of these. We do not know the relative importance of the different carbohydrate chains that are potentially involved or which sugar residues are likely to be crucial. To address these issues we have carried out a number of studies on the 2G12-gp120 interaction. These include a detailed glycan analysis of the gp120 N-linked carbohydrates, an examination of the effects of digestion of gp120 by various glycosidases, an analysis of the ability of various glycans and lectins to inhibit the interaction, extensive alanine scanning mutagenesis of gp120, sequence comparisons of gp120s with different abilities to interact with 2G12 and, finally, modeling studies of gp120. We conclude that Manα1→2Man-linked residues of the outer face of gp120 are required for the 2G12 epitope. Extensive site-directed mutagenesis demonstrated little dependence of 2G12 affinity on specific gp120 amino acid side chains.

Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

Abstract: The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

Herpesvirus Assembly and Egress

Abstract: Herpesvirus particles consist of four morphologically distinct structures, the core, capsid, tegument, and envelope. The inner nucleoprotein core comprising the linear double-stranded DNA genome is included in an icosahedral (T=16) capsid shell of 150 hexons and 12 pentons. The capsid is surrounded by a layer of proteinaceous material designated the tegument which, in turn, is enclosed in an envelope of host cell-derived lipids containing virus-encoded (glyco)proteins. Whereas capsid formation in the nuclei of infected cells is understood in some detail, the mechanisms of tegumentation and envelopment and the intracellular compartments involved have long been disputed. This review focuses on recent findings that demonstrate a rather complex process of herpesvirus maturation including primary envelopment of capsids by budding at the inner leaflet of the nuclear membrane and translocation of capsids into the cytoplasm after loss of the primary envelope by fusion with the outer leaflet of the nuclear membrane. Subsequently, final tegumentation occurs in the cytoplasm and tegumented capsids obtain their final envelope by budding into vesicles of the trans-Golgi network. Tegumentation and envelopment are driven by specific protein-protein interactions that appear, at least in cultured cells, to exhibit a remarkable redundancy.

C-Type Lectins DC-SIGN and L-SIGN Mediate Cellular Entry by Ebola Virus in cis and in trans

Abstract: Ebola virus is a highly lethal pathogen responsible for several outbreaks of hemorrhagic fever. Here we show that the primate lentiviral binding C-type lectins DC-SIGN and L-SIGN act as cofactors for cellular entry by Ebola virus. Furthermore, DC-SIGN on the surface of dendritic cells is able to function as a trans receptor, binding Ebola virus-pseudotyped lentiviral particles and transmitting infection to susceptible cells. Our data underscore a role for DC-SIGN and L-SIGN in the infective process and pathogenicity of Ebola virus infection.

The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120.

Abstract: We have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.

Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes.

Abstract: Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-β enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology

Abstract: Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.

Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling

Abstract: In the course of acute measles, an efficient virus-specific immune response is generated which mediates viral clearance from the host and confers protection against reinfection. Paradoxically, a general immunosuppression is also induced favoring secondary infections, which are the major reason for the annual high morbidity and mortality rates associated with measles. The magnitude and duration of immune activation and immune suppression differ between natural or experimental infection and vaccination (20, 60). Studies addressing measles virus (MV)-induced immune suppression mainly have focused on alterations of T-cell functions and viability as a consequence of direct MV infection or contact-mediated signaling (53). In vitro observations suggest that MV infection also enhances apoptosis of monocytes and dendritic cells (DC) and affects their antigen-presenting capacity and cytokine release (31, 53). MV interaction with DC and monocytes is, however, also associated with their maturation or activation, respectively, and thus is important for induction of virus-specific immune responses (32, 39, 45, 54, 56). Strains expressing an MV wild-type-derived hemagglutinin (H) protein reveal a particular tropism for DC and are more efficient in inducing both DC maturation and immunosuppression (32, 48, 54). The mechanisms by which MV leads to these functional alterations are largely unknown. Downregulation of interleukin-12 (IL-12) production in monocytes was linked to MV- or antibody-mediated cross-linking of CD46, the receptor for certain MV strains (29). Lymphotropic MV wild-type strains and clinical isolates, with few known exceptions (43), fail to interact with CD46 but require CD150 for cell entry (15, 26, 49, 59). This molecule is absent from unstimulated monocytes and immature DC (33, 45, 48), and it is thus unknown how infection of these cells by CD150-dependent MV strains occurs. Mammalian Toll-like receptors (TLRs) were implicated in the innate immune recognition of a variety of bacterial pathogens and bacterial products (2). Ten TLR family members were discovered, and several of these appear to play important roles in the activation of cells by various bacterial products. TLR2 is responsible for recognition of gram-positive bacteria (57, 65), bacterial lipoproteins (12, 42), and lipoteichoic acid (38, 55). TLR4 appears to be the main receptor for lipopolysaccharide (LPS) lipid A from gram-negative bacteria (41), TLR6 might be a coreceptor for TLR2 in recognizing certain bacterial structures (50, 58), and TLR9 and TLR3 mediate responses to CpG bacterial DNA and double-stranded RNA (dsRNA), respectively (3, 24). Hence, these receptors are able to discriminate between different bacteria and bacterial structures and thereby direct a proper immune response to a specific pathogen. Intracellular domains of the TLRs share motifs with the protein families of the IL-1 receptors, and a common intracellular pathway leading to activation of NF-κB and mitogen-activated protein kinases involves MyD88, IRAK, and TRAF6 (2). However, other signaling pathways upstream of NF-κB have been described which also include utilization of the phosphatidylinositol-3/Akt-kinase pathway by TLR2 (4). It has recently been demonstrated that mammalian TLR signaling can also be regulated by viral gene products. Vaccinia virus encodes gene products that interfere with proximal TLR signaling in the cytoplasm (11), and the fusion protein of respiratory syncytial virus (RSV) was found to activate cells via TLR4 and CD14 (35). Using CHO cells stably overexpressing TLR2 or TLR4, we found that MV wild-type strains specifically activated cells via TLR2, and this was dependent on the expression of the H protein of the MV wild-type strain, WTF. The failure of attenuated MV strains to induce TLR2 activation correlated with a single amino acid exchange at position 481 which is, in turn, essential for the usage of CD46 as receptor by these strains. Importantly, MV expressing the wild-type H protein induced activation of TLR-responsive genes such as IL-1α/β, IL-6, and IL-12 p40 in monocytes and also the expression of CD150, the receptor for all MV strains. Activation of TLR signaling by wild-type MV H protein may thus essentially contribute to the immune activation during measles, and loss of the capability to activate TLR2 may be considered as an attenuation marker.

Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication

Abstract: Virions of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses contain conical cores consisting of a protein shell composed of the viral capsid protein (CA) surrounding an internal viral ribonucleoprotein complex. Although genetic studies have implicated CA in both early and late stages of the virus replication cycle, the mechanism of core disassembly following penetration of target cells remains undefined. Using quantitative assays for analyzing HIV-1 core stability in vitro, we identified point mutations in CA that either reduce or increase the stability of the HIV-1 core without impairing conical core formation in virions. Alterations in core stability resulted in severely attenuated HIV-1 replication and impaired reverse transcription in target cells with only minimal effects on viral DNA synthesis in permeabilized virions in vitro. We conclude that formation of a viral core of optimal stability is a prerequisite for efficient HIV-1 infection and suggest that disassembly of the HIV-1 core is a regulated step in infection that may be an attractive target for pharmacologic intervention.

Stabilization of the Soluble, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1

Abstract: The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer (N. Schulke, M. S. Vesanen, R. W. Sanders, P. Zhu, D. J. Anselma, A. R. Villa, P. W. H. I. Parren, J. M. Binley, K. H. Roux, P. J. Maddon, J. P. Moore, and W. C. Olson, J. Virol. 76:7760-7776, 2002). Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.

The Complementary Strand of the Human T-Cell Leukemia Virus Type 1 RNA Genome Encodes a bZIP Transcription Factor That Down-Regulates Viral Transcription

Abstract: The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral trans-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.

Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference

Abstract: Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.

Selectable Subgenomic and Genome-Length Dicistronic RNAs Derived from an Infectious Molecular Clone of the HCV-N Strain of Hepatitis C Virus Replicate Efficiently in Cultured Huh7 Cells

Abstract: Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.

RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

Abstract: The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain

Abstract: After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Viral Late Domains

Abstract: Domains have been identified in the Gag proteins of a number of retroviruses, and in the matrix proteins of the rhabdoviruses and filoviruses, that play a critical role in the pinching off of virus particles from the plasma membrane. These sequences are collectively termed late or L domains to reflect their function late in the virus budding process. One of the intriguing features of these L domains is that they all contain highly conserved motifs known to mediate protein-protein interactions between cellular proteins. Three classes of motifs have been defined in viral L domains: PTAP, PPXY, and YXXL. In each case, the integrity of these motifs appears to be essential for L domain activity, suggesting that L domains function by interacting with a host factor(s). This minireview summarizes the present state of our knowledge concerning viral L domain function and describes recent tantalizing clues regarding the identity of the cellular partners with which L domains interact.

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Abstract: The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

Differential Activation of Innate Immune Responses by Adenovirus and Adeno-Associated Virus Vectors

Abstract: Gene therapy holds promise for the treatment of many inherited and acquired human diseases. Despite numerous early clinical trials, the effectiveness of human gene transfer and expression remains disappointingly low due to limitations in the current generation of vectors (36). The ideal vector for human application should permit targeted, highly efficient, long-term gene delivery and be devoid of significant toxicity (26, 36). Adenovirus vectors have several advantages. Recombinant adenoviruses are easy to manipulate and produce in high titer, can package large quantities of DNA, and have a broad cell tropism. Potent host inflammatory and immune responses, however, limit adenovirus vectors, resulting in transient gene expression and significant morbidity in vivo (17). Adeno-associated virus (AAV) vectors are increasingly being developed for human gene therapy. AAV vectors demonstrate long-term gene expression in vivo, a broad host range, and the ability to infect growth-arrested cells. In contrast to adenovirus vectors, AAV vectors have not been associated with acute inflammation (7, 8, 17). Since the use of AAV in human gene therapy protocols is on the rise, it is important to understand the biology of the host response to these vectors. The acute inflammation induced by adenovirus vectors is due to the activation of target cells and the innate immune system (5, 32). It was previously demonstrated that adenovirus vectors induce the expression of chemokines following transduction in epithelial cells in vitro (5, 27). The induction of chemokines in these target cells is direct and occurs independently of virus transcription. In vivo, adenovirus vectors also activate the innate immune system (32, 40). The innate immune system is comprised of numerous cellular components including neutrophils, natural killer cells, and macrophages that recognize pathogens according to the pattern of their surface components rather than their specific antigenic sequences (14). An invading pathogen induces a cell to respond rapidly (within minutes to hours) and release an array of inflammatory mediators including cytokines and chemokines (31). The activation of innate immune responses in vivo also occurs in the absence of virus gene transcription (24, 32). The activation of target cells and the innate immune system is an important component of host recognition and response to adenovirus vectors (14). The direct activation of target cells and the innate immune system by psoralen-inactivated adenovirus vectors suggests a role for the viral capsid in these responses. Thus, the differential induction of acute inflammation in vivo arising from adenovirus and AAV infection is not clear given the similar features of both vectors. Adenoviridae contain a linear double-stranded DNA genome, which is encapsulated in an icosahedral protein shell that is approximately 70 nm in diameter (17). Adenovirus particles bind to the 46-kDa coxsackievirus adenovirus receptor and are internalized by receptor-mediated endocytosis in clathrin-coated vesicles. αv integrins (αvβ1, αvβ3, αvβ5) are essential for the efficient internalization of adenovirus vectors (21, 28, 38). The AAV genome is encapsulated as a single-stranded DNA molecule. The nonenveloped virion is also icosahedral in shape but measures only 20 to 25 nm in diameter (7). Initial attachment of AAV to cells is mediated by interaction with heparan sulfate proteoglycan (34). Fibroblast growth factor receptor 1 has also been implicated in virus attachment (29). Using αvβ5 integrins as a coreceptor, AAV vectors gain entry into cells via receptor-mediated endocytosis in a manner similar to that of adenoviruses (2, 33). The objective of this study is to determine the effects of AAV vector transduction on the host chemokine induction in vitro and on the activation of innate immune responses in vivo. We demonstrate that adenovirus but not AAV vectors induce chemokine expression in target cells at equivalent titers and transduction efficiencies in vitro. Furthermore, we show for the first time that AAV vectors transiently activate innate immune responses in vivo. These results increase our understanding of viral vector biology and provide a basis for the reduced inflammatory properties exhibited by AAV vectors.

Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 and Its Isolated RING Finger Domain Act as Ubiquitin E3 Ligases In Vitro

Abstract: Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.

Influenza Virus Can Enter and Infect Cells in the Absence of Clathrin-Mediated Endocytosis

Abstract: Influenza virus has been described to enter host cells via clathrin-mediated endocytosis. However, it has also been suggested that other endocytic routes may provide additional entry pathways. Here we show that influenza virus may enter and infect HeLa cells that are unable to take up ligands by clathrin-mediated endocytosis. By overexpressing a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis, we demonstrate that while transferrin uptake and Semliki Forest virus infection were prevented, influenza virus could enter and infect cells expressing Eps15Δ95/295. This finding is supported by the successful infection of cells with influenza virus in the presence of chemical treatments that block endocytosis, namely, chlorpromazine and potassium depletion. We show also that influenza virus may infect cells incapable of uptake by caveolae. Treatment with the inhibitors nystatin, methyl-β-cyclodextrin, and genistein, as well as transfection of cells with dominant-negative caveolin-1, had no effect on influenza virus infection. By combining inhibitory methods to block both clathrin-mediated endocytosis and uptake by caveolae in the same cell, we demonstrate that influenza virus may infect cells by an additional non-clathrin-dependent, non-caveola-dependent endocytic pathway. We believe this to be the first conclusive analysis of virus entry via such a non-clathrin-dependent pathway, in addition to the traditional clathrin-dependent route.

Magnitude of Functional CD8+ T-Cell Responses to the Gag Protein of Human Immunodeficiency Virus Type 1 Correlates Inversely with Viral Load in Plasma

Abstract: The importance of CD8 T-cell responses in the control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated, yet few studies have been able to correlate these responses with markers of HIV-1 disease progression. This study measured cell-mediated immune responses using peripheral blood mononuclear cells (PBMC) obtained from 27 patients with chronic HIV-1 infection, the majority of whom were off antiretroviral therapy. The ELISPOT assay was used to detect gamma interferon-secreting PBMC after stimulation with overlapping HIV-1 peptides spanning the Gag, Pol, Env, and Nef proteins in addition to the baculovirus-derived p24 and gp160 proteins. All volunteers had responses to at least one HIV-1-specific peptide. All but one of the subjects (96%) responded to the Gag peptide pool, and 86% responded to the Pol and/or Nef peptide pools. The magnitude and the breadth of T-cell responses directed to either the Gag or p24 peptide pools correlated inversely with viral load in plasma (r 0.60, P < 0.001 and r 0.52, P < 0.005, respectively) and directly with absolute CD4 T-cell counts (r 0.54, P < 0.01 and r 0.39, P < 0.05, respectively) using the Spearman rank correlation test. Responses to the Pol and integrase peptide pools also correlated with absolute CD4 T-cell counts (r 0.45, P < 0.05 and r 0.49, P < 0.01, respectively). No correlation with markers of disease progression was seen with specific T-cell responses directed toward the Env or Nef peptides. These data serve as strong evidence that major histocompatibility complex class I presentation of Gag peptides is an essential feature for any HIV-1 vaccine designed to elicit optimal CD8 T-cell responses.

Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides

Abstract: We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage φ6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the φ6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.

Pathogenesis of avian influenza A (H5N1) viruses in ferrets.

Abstract: Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 107 50% egg infectious doses (EID50) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 107 EID50 of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.

Ebola Virus VP40 Drives the Formation of Virus-Like Filamentous Particles Along with GP

Abstract: Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.

Human Immunodeficiency Virus Type 1 Uses Lipid Raft-Colocalized CD4 and Chemokine Receptors for Productive Entry into CD4+ T Cells

Abstract: In this report, we describe a crucial role of lipid raft-colocalized receptors in the entry of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T cells. We show that biochemically isolated detergent-resistant fractions have characteristics of lipid rafts. Lipid raft integrity was required for productive HIV-1 entry as determined by (i) semiquantitative PCR analysis and (ii) single-cycle infectivity assay using HIV-1 expressing the luciferase reporter gene and pseudotyped with HIV-1 HXB2 envelope or vesicular stomatitis virus envelope glycoprotein (VSV-G). Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) relocalized raft-resident markers to a nonraft environment but did not significantly change the surface expression of HIV-1 receptors. MbetaCD treatment inhibited productive infection of HIV-1 by 95% as determined by luciferase activity in cells infected with HXB2 envelope-pseudotyped virus. In contrast, infection with VSV-G-pseudotyped virus, which enters the cells through an endocytic pathway, was not suppressed. Biochemical fractionation and confocal imaging of HIV-1 receptor distribution in live cells demonstrated that CD4, CCR5, and CXCR4 colocalized with raft-resident markers, ganglioside GM1, and glycosylphosphatidylinositol-anchored CD48. While confocal microscopy analysis revealed that HIV-1 receptors localized most likely to the same lipid microdomains, sucrose gradient analysis of the receptor localization showed that, in contrast to CD4 and CCR5, CXCR4 was associated preferentially with the nonraft membrane fraction. The binding of HIV-1 envelope gp120 to lipid rafts in the presence, but not in the absence, of cholesterol strongly supports our hypothesis that raft-colocalized receptors are directly involved in virus entry. Dramatic changes in lipid raft and HIV-1 receptor redistribution were observed upon binding of HIV-1 NL4-3 to PM1 T cells. Colocalization of CCR5 with GM1 and gp120 upon engagement of CD4 and CXCR4 by HIV-1 further supports our observation that HIV-1 receptors localize to the same lipid rafts in PM1 T cells.

Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids.

Abstract: The wild-type UL31, UL34, and US3 proteins localized on nuclear membranes and perinuclear virions; the US3 protein was also on cytoplasmic membranes and extranuclear virions. The UL31 and UL34 proteins were not detected in extracellular virions. US3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the US3 protein plays an important, but nonessential, role in the egress pathway.

A Natural Intergenotypic Recombinant of Hepatitis C Virus Identified in St. Petersburg

Abstract: Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5' untranslated region (5'UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5'UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the city's population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1-2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.