Showing papers in "Journal of Virology in 2003"

Viral Persistence Alters CD8 T-Cell Immunodominance and Tissue Distribution and Results in Distinct Stages of Functional Impairment

Abstract: Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.

The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex

Abstract: Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.

Severe CD4+ T-Cell Depletion in Gut Lymphoid Tissue during Primary Human Immunodeficiency Virus Type 1 Infection and Substantial Delay in Restoration following Highly Active Antiretroviral Therapy

Abstract: Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4 + T-cell depletion and the effect of HAART on the restoration of CD4 + T cells in GALT. Severe depletion of intestinal CD4 + T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4 + T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4 + T cells, despite the delay in comparison to peripheral blood CD4 + T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4 + T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4 + T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4 + T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.

CD8+ T Cells Mediate Viral Clearance and Disease Pathogenesis during Acute Hepatitis B Virus Infection

Abstract: Although the CD4 + - and CD8 + -T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8 + cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8 + -T-cell response.

Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference

Abstract: RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

Comprehensive Epitope Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Responses Directed against the Entire Expressed HIV-1 Genome Demonstrate Broadly Directed Responses, but No Correlation to Viral Load

Abstract: Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Human Cytomegalovirus Activates Inflammatory Cytokine Responses via CD14 and Toll-Like Receptor 2

Abstract: Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen. Clinically, CMV disease correlates with immune suppression in which severe presentations are evident in neonates, persons with AIDS, and other immune-suppressed patient groups (38). CMV infection of neonates is associated with deafness, mental retardation, and mortality, whereas AIDS patients often suffer a blinding CMV retinitis, as well as pneumonia and gastrointestinal inflammation. In organ transplant recipients, a patient group hard hit by CMV infection, disease is associated with an increased frequency of graft rejection and is a major cause of posttransplant infection. The varied array of clinical disease correlates with the exceptionally broad tropism of this virus. Indeed, histological analysis of autopsy tissues obtained from patients with CMV disease has demonstrated infected cells in virtually all organs. At the cellular level, CMV can infect monocytes/macrophages, endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, stromal cells, neuronal cells, neutrophils, and hepatocyes (13, 17, 35, 44, 48, 49, 61). In fact, CMV is a suspected pathogenetic agent in cardiovascular disease due to its ability to persist in large-vessel endothelial cells and to infect all cell types involved in cardiovascular lesions (24). Cells exposed to CMV undergo a number of physiological changes that are rendered upon the cell with extremely rapid kinetics (15). These events include changes in Ca2+ homeostasis (1) and activation of phospholipase C and phospholipase A2, as well as increased release of arachidonic acid and its metabolites (1, 59). All of these changes can be triggered by UV-inactivated virions, suggesting that structural components of the virus are responsible for the alterations in cell physiology and intracellular signaling that occur during virus-cell contact and/or virus entry. Virus-cell contact also results in the activation of the transcription factors NF-κB and SP-1, as well as mitogen-activated protein (MAP) kinase, ERK1/2, and p38 (7, 26, 63). Activation of transcription factors by CMV suggests that alterations in cellular transcription should occur in CMV-infected cells, and this is precisely the case. Several transcriptional-profiling studies reveal that cells infected with CMV exhibit profound reprogramming of gene expression (8, 47, 64, 65). Interestingly, the most strongly induced genes were indicators of innate immune activation. Antiviral genes belonging to the interferon-stimulated gene family (ISGs) and inflammatory genes, such as those for RANTES, interleukin 6 (IL-6), IL-7, IL-11, and cyclooxygenase 2 (COX-2), were all robustly induced in CMV-infected fibroblasts (8, 47, 64, 65). Induction of these innate immune markers did not require virus replication. Indeed, cells treated with only the primary ligand of CMV, glycoprotein B (gB) (6), exhibited a response very similar, but not wholly identical, to that of cells treated with intact virus (47). In particular, cells treated with gB strongly induced ISGs. Taken together, the findings suggest that a signal transduction pathway is activated by cell contact of CMV envelope proteins, resulting in numerous physiological changes that culminate, in part, with innate immune activation. The innate immune system is an ancient, universal host defense system. A limited number of evolutionarily conserved germ line receptors found in plants, Drosophila, and humans mediate certain innate immune responses. Termed pattern recognition receptors, these molecules form the basis of the primary host alarm system in response to pathogen-associated molecular patterns (PAMPs) (23). Toll-like receptors (TLRs) are now understood to play a major role in pathogen recognition. Stimulation of TLRs by pathogens activates signal transduction pathways that lead to induction of a range of antimicrobial genes and inflammatory cytokines (2, 23, 29). In addition, key costimulatory molecules, such as CD80 and CD86, which are important for activation of adaptive immunity, are also induced as a consequence of TLR signaling. At present, 10 TLR molecules have been described in humans and mice. Ligands ranging from lipopolysaccharide (LPS) of gram-negative bacteria, peptidoglycan of gram-positive bacteria, flagellin, CpG DNA, and various components from mycobacteria, yeast, and parasitic pathogens, are all detected by TLRs. Although the precise molecular patterns are not completely characterized, PAMPs are hypothesized to be macromolecular modifications unique to these organisms. Viruses have long been known to activate innate immune responses characterized by the induction of inflammatory cytokines and a comprehensive set of ISGs (45, 53). Until recently, the only identified molecular trigger of host innate responses was double-stranded RNA, a common replicative intermediate in the life cycle of many viruses. Double-stranded RNA can activate a key interferon transcription factor, interferon regulatory factor 3 (IRF-3), and induce synthesis of many antiviral genes. Generally, however, the mechanisms by which viruses activate innate immunity remain largely undefined. Since viruses are obligate intracellular parasites, virus-encoded proteins are synthesized by host machinery and ultimately bear protein modifications reflective of the host. Thus, it is not immediately obvious what PAMPs are displayed on viruses. Currently, however, data are clearly emerging showing that TLRs detect viruses and trigger inflammatory responses. Respiratory syncytcial virus (RSV) and mouse mammary tumor virus (MMTV) both signal through TLR4 (28, 42), the well-described LPS receptor. In the case of RSV, TLR4-deficient mice challenged with RSV exhibited a variety of impaired innate immune functions and an inability to clear the virus (20). These findings strongly suggest that Toll signaling pathways have an important role in the innate immunity to RSV. More recently, measles virus was reported to be detected by TLR2, a TLR with broad ligand recognition properties (5). The most striking common feature of these first three reports of virus detection by TLRs is that TLR responses were triggered by viral envelope glycoproteins. Specifically, the fusion protein of RSV, the envelope (env) protein of MMTV, and the hemagglutinin protein of measles virus were the identified triggers of the TLR responsiveness in their respective systems. Interestingly, all of these proteins play key roles in the virus entry pathway, such as attachment and fusion. These studies plainly point to a heretofore-unknown ability of the host to detect viruses during entry but prior to the onset of any replication events. Here, we report that TLR2 and CD14 form the central basis of the membrane detection machinery for CMV.

Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harboring Subgenomic Replicons

Abstract: Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5'-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.

Herpesvirus entry: an update.

Abstract: The entry of herpesviruses into cells depends upon interactions of several viral glycoproteins with multiple cell surface receptors. Also, each herpesvirus may have evolved multiple pathways for entry into different cell types, as is evident for HSV and EBV. The broad host range of HSV is consistent with its use of cell surface heparan sulfate as a binding receptor and both 3-O-sulfated heparan sulfate and multiple conserved and widely expressed proteins as entry receptors. The more limited host range of EBV (at least in the case of its B-cell target) is also consistent with its use of binding and entry receptors that are found together on very few cell types, chief of which are B cells. Much remains to be learned about the actual requirements for entry of these viruses into the target cell types that are critical for disease and about the mechanisms of virus-induced membrane fusion.

Host Factors in Positive-Strand RNA Virus Genome Replication

Abstract: All viruses are gene poor relative to their hosts: even the largest viral genomes only encode hundreds of genes, while those of host cells typically encode tens of thousands of genes. Thus, most steps in virus infection involve interactions between relatively few different types of viral components and much more complex pools of host factors. This sea of host factors represents both the essential milieu to which viruses must adapt for survival and a tremendous, manipulatable resource for gene-poor viruses. Accordingly, host factors play important roles in most steps of viral infection, and identifying such host factors and their contributions has long been recognized as an important frontier. The continuing emergence of close integration between viral and host functions in infection suggests moving beyond separate views of virus and host to a more holistic view of the virus-infected cell as a unified entity that constitutes the functional unit of infection. One area in which the importance of host factors is increasingly emerging is the replication of positive-strand RNA viruses. Positive-strand RNA viruses encompass over one-third of all virus genera and include numerous pathogens, such as the severe acute respiratory syndrome coronavirus SARS, hepatitis C virus (HCV), and many of the viruses on the U.S. Health and Human Services Department Select List of potential bioterrorism agents. Host factors participate in most, if not all, steps of positive-strand RNA virus infection, including entry, viral gene expression, virion assembly, and release. Moreover, host factors are targeted by positive-strand RNA viruses to modulate host gene expression and defenses. This review focuses on host factors involved in positivestrand RNA virus genome replication. The evidence for such host factor involvement has come from varied genetic and biochemical approaches (15). These include, among others, studies based on the varying permissiveness of some cell types and extracts for RNA replication (2, 3, 4, 19); identification of many host proteins that interact with viral genomic RNAs or replication proteins and, in some cases, have been functionally linked to replication (5, 18, 27, 35, 37, 40); and mutational screens in genetic model systems, such as Arabidopsis thaliana (17, 39) and the yeast Saccharomyces cerevisiae (8, 11, 16, 23, 38). Recent data show that host factors play important roles in assembling the viral RNA replication complex, selecting and recruiting viral RNA replication templates, activating the complex for RNA synthesis, and other steps. Each of these virushost interactions may contribute to the host specificity, tissue specificity, or pathology of infections. Each such virus-host interaction also represents a potential target for virus control or for optimization to improve beneficial uses of viruses and their components. Positive-strand RNA viruses can be divided into a number of superfamilies defined by distinguishable RNA replication genes and features (41). Nevertheless, the RNA replication mechanisms of these viruses share sufficient similarities to make it reasonable to discuss their replication as a class and to attempt to draw general lessons by comparing examples from different superfamilies. As discussed further below, a few of the common features shared by positive-strand RNA viruses are the need to coordinate use of the infecting viral genomic RNA as a template for translation and replication, assembly of replication complexes on intracellular membranes, and production of 10- to 100-fold excesses of positive- over negativestrand RNA. A simplified general scheme for RNA replication by positive-strand RNA viruses is shown in Fig. 1. Below we discuss the involvement of host factors in the various stages of this RNA replication process, using examples from a number of viruses. We regret that space limitations do not allow us to cite all of the work being done in this exciting area.

Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity

Abstract: Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5. Chemicals/CAS: Adenovirus E1B Proteins; Antibodies, Viral

Improved Coreceptor Usage Prediction and Genotypic Monitoring of R5-to-X4 Transition by Motif Analysis of Human Immunodeficiency Virus Type 1 env V3 Loop Sequences

Abstract: Early studies of the biological properties of human immunodeficiency virus type 1 (HIV-1) found that virus isolates could be placed into as few as two phenotypic categories (defined in vitro as either non-syncytium-inducing [NSI] or syncytium-inducing [SI]) in certain CD4+ T-cell lines. These phenotypes were often found to be associated with differences in growth properties and cytopathicity on peripheral blood mononuclear cells (PBMC) (1, 14, 46) and in cellular host range (3, 48). Ultimately, the difference between the NSI and SI phenotypes was shown to be due largely to the differential use of chemokine receptors as coreceptors for viral entry: NSI viruses predominantly use CCR5, while SI viruses can use CCR5 and CXCR4 or CXCR4 exclusively (2, 29, 31, 52, 54). Results determined on the basis of SI phenotype and/or coreceptor usage typing showed that although HIV-1 present at primary infections used the CCR5 coreceptor (R5 virus) ∼90% of the time (63, 67, 68), a substantial proportion of individuals eventually developed virus that used the CXCR4 coreceptor (X4 virus). These X4/SI viruses are associated with accelerated CD4 decline and more rapid progression of HIV-1 disease (8, 28, 33, 43, 47). Little is known about the mechanisms by which these viruses come to predominate among the HIV-1 strains present in an infected person. For example, it is not known whether X4 emergence is a primary pathogenic event or is secondary to some other event, i.e., whether the virus itself causes accelerated disease progression or whether another event causes the acceleration and perhaps also leads to X4 outgrowth. Another important unanswered question is whether X4 viruses arise multiple times during the course of disease and, if so, why they do not become dominant whenever they emerge. There is also uncertainty about the frequency with which phenotype transition occurs. Phenotypic studies suggest that 50 to 60% of progressing subjects acquire X4/SI virus (26, 57, 58), but the results of a detailed longitudinal genotypic study have indicated the occurrence, sometimes transient, of at least one of four X4-associated mutations in nine of nine individuals (50). Coreceptor usage of a particular virus is established by functional assays (growth on MT2 cells [28] or infection of indicator cell lines [64]). These assays are limited, however, in that results are generally reported only as positive or negative and provide no insight into the sequence of mutations responsible for the phenotype switch—information which may further clarify the role of X4 viruses in pathogenesis, as we discuss below. Certain mutations, particularly in the V3 loop of env (5-7, 15, 16, 22, 23, 51), are strongly associated with syncytium induction and CXCR4 usage; in particular, basic amino acids at V3 positions 11 and 25 (amino acid coordinates 306 and 322 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"K03455","term_id":"1906382","term_text":"K03455"}}K03455] in standard reference HXB2) very frequently distinguish primary X4 from R5 viruses (9, 15, 16, 21, 42, 66), with positions 24 and 27 implicated in some cases (10, 37). However, while evolutionary studies of the R5-X4 transition have been undertaken (30, 38, 61, 62), the actual mutational pathway or pathways by which R5 viruses that establish infections in vivo evolve into X4 viruses (i.e., the specific evolutionary sequence of mutations required) has been largely unexplored. It is not clear whether the appearance of basic amino acids at sites 11 and 25 is sufficient or even necessary in most cases in vivo to lead to the outgrowth of X4 virus or whether, instead, a more gradual process of mutation accumulation takes place. If mutant accumulation does occur in a more gradual process, this may provide an opportunity for both early detection and arrest of X4 development. Viruses that can use both coreceptors (R5X4 viruses) are known to arise around the time of R5-to-X4 transition (2, 8, 38, 61), but their evolutionary role in that transition is not certain. The answers to questions of in vivo evolution cannot be approached by phenotypic assays alone, and analyses based on the appearance of positively charged mutations at V3 sites 11 and 25 have led to incomplete and sometimes ambiguous conclusions regarding the transition process (35). The V3 loop is highly variable within and between individuals, and bioinformatic approaches suggest that many changes not yet examined virologically are likely to influence coreceptor usage (21, 24, 42). To gain a broader understanding of the mutations that contribute to X4 phenotype and of the temporal sequence in which they occur, we used position-specific scoring matrices (PSSM) (19, 20) to analyze V3 sequences. PSSM are used to detect nonrandom distributions of amino acids at adjacent sites associated with empirically determined groupings of sequences. They are frequently used to search DNA or protein sequences for particular motifs, e.g., transcriptional regulatory sites (55), coiled-coil domains (32), major histocompatibility complex class I binding sites (41), and others. A PSSM uses background genetic variation as a baseline comparison, or “null model,” to facilitate comparison of the residues of a sequence fragment to those of a group of aligned sequences known to have the desired property. The comparison leads to a score that can be interpreted as indicating the likelihood that the sequence fragment has the property of interest. In our study, the empirical groupings consist of V3 loop sequences associated with X4 (or SI) virus and R5 (or NSI) viruses. Using the PSSM as described below, a sequence can be assigned a score: the higher the score, the more closely the sequence resembles those of known X4 viruses. We used the PSSM score for two purposes. First, we developed a PSSM-based phenotype predictor usable for all V3 sequences. We explored the statistical properties of this predictor and showed that it outperforms simple methods that categorize sequences on the basis of the presence of basic amino acids at sites 11 or 25. We validated the predictor with two sets of V3 sequences from phenotyped viruses different from those used to produce the PSSM matrix. Second, we showed that the score can serve as a measure of the transition from R5 to X4 phenotype. Since the PSSM score can act as a continuous indicator of X4 evolution, we used it to identify common temporal patterns among 11 serially sampled individuals. By scoring reconstructed ancestors of the sampled virus for each subject, we demonstrated that the progression from low-scoring (R5-like) to high-scoring (X4-like) viruses was generally gradual but that the loss of putative X4 virus at the later stages of infection can occur in two different ways.

B Cells and Antibody Play Critical Roles in the Immediate Defense of Disseminated Infection by West Nile Encephalitis Virus

Abstract: West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (μMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, a ∼500-fold difference in serum viral load was detected in μMT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected μMT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.

The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

Abstract: The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation.

Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

Abstract: Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.

Origin and Evolution of Japanese Encephalitis Virus in Southeast Asia

Abstract: Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene

Abstract: Cellular immune responses, particularly those associated with CD3 CD8 cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4 and CD8 T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Human Immunodeficiency Virus Type 1 Escape from RNA Interference

Abstract: Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.

Functional Surfaces of the Human Immunodeficiency Virus Type 1 Capsid Protein

Abstract: The human immunodeficiency virus type 1 initially assembles and buds as an immature particle that is organized by the viral Gag polyprotein. Gag is then proteolyzed to produce the smaller capsid protein CA, which forms the central conical capsid that surrounds the RNA genome in the mature, infectious virus. To define CA surfaces that function at different stages of the viral life cycle, a total of 48 different alanine-scanning surface mutations in CA were tested for their effects on Gag protein expression, processing, particle production and morphology, capsid assembly, and infectivity. The 27 detrimental mutations fall into three classes: 13 mutations significantly diminished or altered particle production, 9 mutations failed to assemble normal capsids, and 5 mutations supported normal viral assembly but were nevertheless reduced more than 20-fold in infectivity. The locations of the assembly-defective mutations implicate three different CA surfaces in immature particle assembly: one surface encompasses helices 4 to 6 in the CA N-terminal domain (NTD), a second surrounds the crystallographically defined CA dimer interface in the C-terminal domain (CTD), and a third surrounds the loop preceding helix 8 at the base of the CTD. Mature capsid formation required a distinct surface encompassing helices 1 to 3 in the NTD, in good agreement with a recent structural model for the viral capsid. Finally, the identification of replication-defective mutants with normal viral assembly phenotypes indicates that CA also performs important nonstructural functions at early stages of the viral life cycle.

Hepatitis C Virus Glycoproteins Interact with DC-SIGN and DC-SIGNR

Abstract: DC-SIGN and DC-SIGNR are two closely related membrane-associated C-type lectins that bind human immunodeficiency virus (HIV) envelope glycoprotein with high affinity. Binding of HIV to cells expressing DC-SIGN or DC-SIGNR can enhance the efficiency of infection of cells coexpressing the specific HIV receptors. DC-SIGN is expressed on some dendritic cells, while DC-SIGNR is localized to certain endothelial cell populations, including hepatic sinusoidal endothelial cells. We found that soluble versions of the hepatitis C virus (HCV) E2 glycoprotein and retrovirus pseudotypes expressing chimeric forms of both HCV E1 and E2 glycoproteins bound efficiently to DC-SIGN and DC-SIGNR expressed on cell lines and primary human endothelial cells but not to other C-type lectins tested. Soluble E2 bound to immature and mature human monocyte-derived dendritic cells (MDDCs). Binding of E2 to immature MDDCs was dependent on DC-SIGN interactions, while binding to mature MDDCs was partly independent of DC-SIGN, suggesting that other cell surface molecules may mediate HCV glycoprotein interactions. HCV interactions with DC-SIGN and DC-SIGNR may contribute to the establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell function.

Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids

Abstract: Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.

Access of Antibody Molecules to the Conserved Coreceptor Binding Site on Glycoprotein gp120 Is Sterically Restricted on Primary Human Immunodeficiency Virus Type 1

Abstract: Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.

A Novel Antiviral Intervention Results in More Accurate Assessment of Human Immunodeficiency Virus Type 1 Replication Dynamics and T-Cell Decay In Vivo

Abstract: Mathematical models provide an understanding of in vivo replication kinetics of human immunodeficiency virus type 1 (HIV-1). With a novel intervention designed for increased potency, we have more accurately deduced the half-lives of virus-producing CD4(+) T cells, 0.7 day, and the generation time of HIV-1 in vivo, approximately 2 days, confirming the dynamic nature of HIV-1 replication.

Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

Abstract: Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

Identification of Amino Acid Residues in the Capsid Proteins of Adeno-Associated Virus Type 2 That Contribute to Heparan Sulfate Proteoglycan Binding

Abstract: The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.

Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins.

Abstract: We have generated a recombinant Newcastle disease virus (NDV) that expresses the green fluorescence protein (GFP) in infected chicken embryo fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment of cells with chicken alpha/beta IFN (IFN-α/β) completely blocks viral GFP expression. Prior transfection of plasmid DNA induces an IFN response in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors of the IFN-α/β system, including the influenza A virus NS1 protein and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore conclude that the NDV-GFP virus could be used to screen proteins expressed from plasmids for the ability to counteract the host cell IFN response. Using this system, we show that expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-GFP replication in the face of the transfection-induced IFN response. The V and W proteins of Nipah virus, a highly lethal pathogen in humans, also block activation of an IFN-inducible promoter in primate cells. Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical to the amino terminus of Nipah virus W, is sufficient to exert the IFN-antagonist activity. In contrast, the anti-IFN activity of the NDV V protein appears to be located in the carboxy-terminal region of the protein, a region implicated in the IFN-antagonist activity exhibited by the V proteins of mumps virus and human parainfluenza virus type 2.

Roles for Endocytosis and Low pH in Herpes Simplex Virus Entry into HeLa and Chinese Hamster Ovary Cells

Abstract: Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.

Adenovirus Type 11 Uses CD46 as a Cellular Receptor

Abstract: The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.

Ex Vivo Profiling of CD8+-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers

Abstract: Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

Overexpression of the α-2,6-Sialyltransferase in MDCK Cells Increases Influenza Virus Sensitivity to Neuraminidase Inhibitors

Abstract: No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.