Laura J. White

Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein

ABSTRACT Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3′ end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3′ end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.

Structural requirements for the assembly of norwalk virus-like particles

ABSTRACT Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.

Role of Alpha/Beta Interferon in Venezuelan Equine Encephalitis Virus Pathogenesis: Effect of an Attenuating Mutation in the 5′ Untranslated Region

ABSTRACT Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5′ untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-α/β) activity in VEE pathogenesis. In IFN-α/βR−/− mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-α/βR−/− mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-α/β pathway. In cells derived from IFN-α/βR−/− mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-α/β plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-α/β between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-α/β in two separate in vitro assays, suggesting that the increased sensitivity to IFN-α/β plays a major role in the in vivo attenuation of V3043.

Flow Cytometry-Based Assay for Titrating Dengue Virus

ABSTRACT Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.

Differential Induction of Type I Interferon Responses in Myeloid Dendritic Cells by Mosquito and Mammalian-Cell-Derived Alphaviruses

ABSTRACT Dendritic cells (DCs) are an important early target cell for many mosquito-borne viruses, and in many cases mosquito-cell-derived arboviruses more efficiently infect DCs than viruses derived from mammalian cells. However, whether mosquito-cell-derived viruses differ from mammalian-cell-derived viruses in their ability to induce antiviral responses in the infected dendritic cell has not been evaluated. In this report, alphaviruses, which are mosquito-borne viruses that cause diseases ranging from encephalitis to arthritis, were used to determine whether viruses grown in mosquito cells differed from mammalian-cell-derived viruses in their ability to induce type I interferon (IFN) responses in infected primary dendritic cells. Consistent with previous results, mosquito-cell-derived Ross River virus (mos-RRV) and Venezuelan equine encephalitis virus (mos-VEE) exhibited enhanced infection of primary myeloid dendritic cells (mDCs) compared to mammalian-cell-derived virus preparations. However, unlike the mammalian-cell-derived viruses, which induced high levels of type I IFN in the infected mDC cultures, mos-RRV and mos-VEE were poor IFN inducers. Furthermore, the poor IFN induction by mos-RRV contributed to the enhanced infection of mDCs by mos-RRV. These results suggest that the viruses initially delivered by the mosquito vector differ from those generated in subsequent rounds of replication in the host, not just with respect to their ability to infect dendritic cells but also in their ability to induce or inhibit antiviral type I IFN responses. This difference may have an important impact on the mosquito-borne viruss ability to successfully make the transition from the arthropod vector to the vertebrate host.

Effects of PKR/RNase L-Dependent and Alternative Antiviral Pathways on Alphavirus Replication and Pathogenesis

Type I interferons (IFN-alpha/beta) rapidly confer resistance to alphavirus infection in macrophages and dendritic cells (DC) as evidenced by the dramatically increased susceptibility of these cells in mice with the IFNAR1 subunit of the IFN-alpha/beta receptor ablated (IFNAR1-/-). Normal adult mice develop only a subclinical Sindbis virus infection, whereas infected IFNAR1-/- mice rapidly succumb to a fatal disease. Here, we investigated the individual and combined contributions of the two best characterized INF-alpha/beta-mediated antiviral pathways to the control of Sindbis virus replication: (1) the coupled 2-5A synthetase/RNase L pathway and (2) the double-stranded RNA-dependent protein kinase (PKR) pathway. Surprisingly, mice deficient in PKR, RNase L, and Mx-1 (triply-deficient [TD]) developed only subclinical infection. Although the permissivity of cells in lymph nodes draining the inoculation site was increased in the absence of PKR/RNase L, systemic dissemination of the virus infection was restricted by an alternative IFN-alpha/beta receptor-dependent mechanism. In vitro, suppression of early virus protein synthesis and virion production in primary bone marrow-derived dendritic cells (BMDC) was largely dependent on the PKR pathway. However, later in infection virion production was reduced even in the absence of PKR/RNase L by an IFN-alpha/beta receptor-dependent mechanism. Priming of BMDC with IFN-alpha/beta or IFN-gamma resulted in dose-dependent restriction of virus replication, largely independent of PKR and/or RNase L expression.

Venezuelan equine encephalitis virus disrupts STAT1 signaling by distinct mechanisms independent of host shutoff.

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-β and IFN-γ. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-β treatment but did inhibit Jak1 and Jak2 activation in response to IFN-γ, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.

Role of humoral versus cellular responses induced by a protective dengue vaccine candidate.

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.

Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis.