Michael J. Conway

West Nile Virus: Biology, Transmission, and Human Infection

SUMMARY West Nile Virus was introduced into the Western Hemisphere during the late summer of 1999 and has been causing significant and sometimes severe human diseases since that time. This article briefly touches upon the biology of the virus and provides a comprehensive review regarding recent discoveries about virus transmission, virus acquisition, and human infection and disease.

Replication and Assembly of Human Papillomaviruses

Human papillomaviruses (HPVs) are small dsDNA tumor viruses, which are the etiologic agents of most cervical cancers and are associated with a growing percentage of oropharyngeal cancers. The HPV capsid is non-enveloped, having a T=7 icosahedral symmetry formed via the interaction among 72 pentamers of the major capsid protein, L1. The minor capsid protein L2 associates with L1 pentamers, although it is not known if each L1 pentamer contains a single L2 protein. The HPV life cycle strictly adheres to the host cell differentiation program, and as such, native HPV virions are only produced in vivo or in organotypic “raft” culture. Research producing synthetic papillomavirus particles—such as virus-like particles (VLPs), papillomavirus-based gene transfer vectors, known as pseudovirions (PsV), and papillomavirus genome-containing quasivirions (QV)—has bypassed the need for stratifying and differentiating host tissue in viral assembly and has allowed for the rapid analysis of HPV infectivity pathways, transmission, immunogenicity, and viral structure.

The Cigarette Smoke Carcinogen Benzo[a]pyrene Enhances Human Papillomavirus Synthesis

ABSTRACT Epidemiological studies suggest that cigarette smoke carcinogens are cofactors which synergize with human papillomavirus (HPV) to increase the risk of cervical cancer progression. Benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, is detected in the cervical mucus and may interact with HPV. Exposure of cervical cells to high concentrations of BaP resulted in a 10-fold increase in HPV type 31 (HPV31) viral titers, whereas treatment with low concentrations of BaP resulted in an increased number of HPV genome copies but not an increase in virion morphogenesis. BaP exposure also increased HPV16 and HPV18 viral titers. Overall, BaP modulation of the HPV life cycle could potentially enhance viral persistence, host tissue carcinogenesis, and permissiveness for cancer progression.

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

ABSTRACT Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.

Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

ABSTRACT Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease.

Role of the Vector in Arbovirus Transmission.

Many arboviral diseases are uncontrolled, and the viruses that cause them are globally emerging or reemerging pathogens that produce significant disease throughout the world. The increased spread and prevalence of disease are occurring during a period of substantial scientific growth in the vector-borne disease research community. This growth has been supported by advances in genomics and proteomics, and by the ability to genetically alter disease vectors. For the first time, researchers are elucidating the molecular details of vector host-seeking behavior, the susceptibility of disease vectors to arboviruses, the immunological control of infection in disease vectors, and the determinants that facilitate transmission of arboviruses from a vector to a host. These discoveries are facilitating the development of novel strategies to combat arboviral disease, including the release of transgenic mosquitoes harboring dominant lethal genes, the introduction of arbovirus-blocking microbes into mosquito populations, and the development of acquisition- and transmission-blocking therapeutics. Understanding the role of the vector in arbovirus transmission has provided critical practical and theoretical tools to control arboviral disease.

Cross-neutralization potential of native human papillomavirus N-terminal L2 epitopes.

Background Human papillomavirus (HPV) capsids are composed of 72 pentamers of the major capsid protein L1, and an unknown number of L2 minor capsid proteins. An N-terminal “external loop” of L2 contains cross-neutralizing epitopes, and native HPV16 virions extracted from 20-day-old organotypic tissues are neutralized by anti-HPV16 L2 antibodies but virus from 10-day-old cultures are not, suggesting that L2 epitopes are more exposed in mature, 20-day virions. This current study was undertaken to determine whether cross-neutralization of other HPV types is similarly dependent on time of harvest and to screen for the most effective cross-neutralizing epitope in native virions. Methodology and Principal Findings Neutralization assays support that although HPV16 L2 epitopes were only exposed in 20-day virions, HPV31 or HPV18 epitopes behaved differently. Instead, HPV31 and HPV18 L2 epitopes were exposed in 10-day virions and remained so in 20-day virions. In contrast, presumably due to sequence divergence, HPV45 was not cross-neutralized by any of the anti-HPV16 L2 antibodies. We found that the most effective cross-neutralizing antibody was a polyclonal antibody named anti-P56/75 #1, which was raised against a peptide consisting of highly conserved HPV16 L2 amino acids 56 to 75. Conclusions and Significance This is the first study to determine the susceptibility of multiple, native high-risk HPV types to neutralization by L2 antibodies. Multiple anti-L2 antibodies were able to cross-neutralize HPV16, HPV31, and HPV18. Only neutralization of HPV16 depended on the time of tissue harvest. These data should inform attempts to produce a second-generation, L2-based vaccine.

Overlapping and independent structural roles for human papillomavirus type 16 L2 conserved cysteines.

Cryoelectron microscopy images of HPV16 pseudovirions (PsV) depict that each pentamer of L1 can be occluded with a monomer of L2. Further research suggests that an N-terminal external loop of L2 exists, which is the target of neutralizing and cross-neutralizing antibodies. Here we show that N-terminal L2 cysteine residues, Cys22 and Cys28, have overlapping and independent structural roles, which affect both early- and late-stage assembly events. Substitution of either cysteine residue enhances infectivity markedly in comparison to wild-type HPV16. However, only Cys22Ser 20-day virions become nearly as stable as wild type. In addition, Cys22Ser, and Cys22,28Ser 20-day virions have lost their susceptibility to neutralization by anti-L2 antibodies, whereas Cys28Ser 20-day virions remain partially susceptible. These results suggest that Cys28 is necessary for late-stage stabilization of capsids, while Cys22 is necessary for proper display of L2 neutralizing epitopes.

Differentiation-Dependent Interpentameric Disulfide Bond Stabilizes Native Human Papillomavirus Type 16

Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature.

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.