Proteome profiling of polyomavirus nuclear replication centers using iPOND.

Polyomaviruses (PyVs) cause diverse diseases in a variety of mammalian hosts. During the life cycle, PyVs recruit nuclear host factors to viral genomes to facilitate replication and transcription. While host factors involved in DNA replication, DNA damage sensing and repair, and cell cycle regulation have been observed to bind PyV DNA, the complete set of viral and host proteins comprising the PyV replisome remains incompletely characterized. Here, the iPOND-MS technique (Isolation of Proteins on Nascent DNA coupled with Mass Spectrometry) was used to identify the proteome bound to murine PyV (MuPyV) DNA immediately following synthesis and 2 hours post-synthesis. Several novel MuPyV DNA interactors were identified on newly synthesized viral DNA (vDNA), including MCM complex members, DNA primase, DNA polymerase alpha, DNA ligase, and replication factor C. Though displaying partial overlap, the host and viral proteins bound to MuPyV DNA 2 hours post-synthesis lacked many of the replication proteins found on newly synthesized vDNA. These data help distinguish between the host factors critical for MuPyV DNA replication and those involved in downstream processing.IMPORTANCEPolyomaviruses are the causative agents of serious diseases in humans, including progressive multifocal leukoencephalopathy (PML), BK virus nephropathy, and Merkel cell carcinoma. The exact mechanisms by which the virus replicates, and which host cell proteins are required, are incompletely characterized. Identifying the host proteins necessary for efficient viral replication in the cell may reveal targets for downstream targets that may suppress viral replication in vivo.

Stabilization of an Infectious Enveloped Virus by Spray-Drying and Lyophilization.

Enveloped viruses are attractive candidates for use as gene- and immunotherapeutic agents due to their efficacy at infecting host cells and delivering genetic information. They have also been used in vaccines as potent antigens to generate strong immune responses, often requiring fewer doses than other vaccine platforms as well as eliminating the need for adjuvants. However, virus instability in liquid formulations may limit their shelf life and require that these products be transported and stored under stringently controlled temperature conditions, contributing to high cost and limiting patient access. In this work, spray-drying and lyophilization were used to embed an infectious enveloped virus within dry, glassy polysaccharide matrices. No loss of viral titer was observed following either spray-drying (at multiple drying gas temperatures) or lyophilization. Furthermore, viruses embedded in the glassy formulations showed enhanced thermal stability, retaining infectivity after exposure to elevated temperatures as high as 85 °C for up to one hour, and for up to 10 weeks at temperatures as high as 30 °C. In comparison, viruses in liquid formulations lost infectivity within an hour at temperatures above 40 °C, or after incubation at 25 °C for longer periods of time.

Effect of mechanical stresses on viral capsid disruption during droplet formation and drying.

Identification of the mechanisms by which viruses lose activity during droplet formation and drying is of great importance to understanding the spread of infectious diseases by virus-containing respiratory droplets and to developing thermally stable spray dried live or inactivated viral vaccines. In this study, we exposed suspensions of baculovirus, an enveloped virus, to isolated mechanical stresses similar to those experienced during respiratory droplet formation and spray drying: fluid shear forces, osmotic pressure forces, and surface tension forces at interfaces. DNA released from mechanically stressed virions was measured by SYBR Gold staining to quantify viral capsid disruption. Theoretical estimates of the force exerted by fluid shear, osmotic pressures and interfacial tension forces during respiratory droplet formation and spray drying suggest that osmotic and interfacial stresses have greater potential to mechanically destabilize viral capsids than forces associated with shear stresses. Experimental results confirmed that rapid changes in osmotic pressure, such as those associated with drying of virus-containing droplets, caused significant viral capsid disruption, whereas the effect of fluid shear forces was negligible. Surface tension forces were sufficient to provoke DNA release from virions adsorbed at air-water interfaces, but the extent of this disruption was limited by the time required for virions to diffuse to interfaces. These results demonstrate the effect of isolated mechanical stresses on virus particles during droplet formation and drying.

A Single Dose, Thermostable, Trivalent Human Papillomavirus Vaccine Formulated Using Atomic Layer Deposition.

Formulations of human papillomavirus (HPV) 16, 18, and 31 L1 capsomere protein antigens were spray dried to obtain glassy microspheres that were then coated by atomic layer deposition (ALD) with nanometer-thin protective layers of alumina. Spray-drying was used to formulate human papillomavirus (HPV) 16, 18, and 31 L1 capsomere protein antigens within glassy microspheres to which nanoscopic protective layers of alumina were applied using ALD. Suspensions of alumina-coated, capsomere-containing microparticles were administered in a single dose to mice. ALD-deposited alumina coatings provided thermostability and a delayed in vivo release of capsomere antigens, incorporating both a prime and a boost dose in one injection. Total serotype-specific antibody titers as well as neutralizing titers determined from pseudovirus infectivity assays were unaffected by incubation of the ALD-coated vaccines for at 4, 50, or 70 °C for three months prior to administration. In addition, even after incubation for three months at 70 °C, single doses of ALD-coated vaccines produced both higher total antibody responses and higher neutralizing responses than control immunizations that used two doses of conventional liquid formulations stored at 4 °C.

Thermostability of a trivalent, capsomere-based vaccine for human papillomavirus infection.

Currently licensed vaccines require a cold-chain to maintain efficacy. This cold-chain requirement reduces the availability of vaccines in resource-poor areas of the world. Commercially available human papillomavirus (HPV) vaccines protect against the most common HPV types related to cervical cancer; however, their impact is limited in many regions due to cold-chain requirements. The goal of this study was to test the thermostability of an adjuvanted, trivalent HPV L1 capsomere-based vaccine (containing HPV types 16, 18, and 31) that was formulated by using lyophilization to embed the antigens within a solid, glassy matrix. Thermal stabilities were determined by storing the vaccine formulations for 3 months at 50 °C, followed by immunization of BALB/c mice and measurement of antibody responses. Antibody responses to capsomere vaccines formulated with alum were unchanged after storage for 3 months at 50 °C. Neutralizing responses to these vaccines were unchanged by high-temperature storage, and were equivalent to those generated after administration of the commercially available liquid HPV vaccine Gardasil®9.

Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers.

During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells.

Single-administration, thermostable human papillomavirus vaccines prepared with atomic layer deposition technology.

Cold-chain requirements affect worldwide distribution of many vaccines. In addition, vaccines requiring multiple doses impose logistical and financial burdens, as well as patient compliance barriers. To address such limitations, we have developed new technologies to prepare thermostable, single-shot, prime-boost microparticle vaccines. Antigen/adjuvant formulations containing glass-forming polymers and trehalose first are spray-dried to form glassy microparticles that confer thermostability. Atomic layer deposition (ALD) reactions conducted in fluidized beds are then used to coat the microparticles with defined numbers of molecular layers of alumina that modulate the timed release of the internalized antigen and act as adjuvants. We have used a model HPV16 L1 capsomere antigen to evaluate the properties of these technologies. Thermostabilized powders containing HPV16 L1 capsomeres were prepared by spray-drying, coated by ALD with up to 500 molecular layers of alumina, and injected into mice. Antigen distribution was assessed by live-animal IR dye tracking of injected labeled antigen. Antibody responses were measured weekly by ELISA, and neutralizing antibodies were measured by pseudovirus neutralization assays at selected time points. Thermostability was evaluated by measuring antibody responses after incubating ALD-coated antigen powders for one month at 50 °C. Single doses of the ALD-coated vaccine formulations elicited a prime-boost immune response, and produced neutralizing responses and antibody titers that were equivalent or superior to conventional prime-boost doses of liquid formulations. Antibody titers were unaffected by month-long incubation of the formulations at 50 °C. Single-dose, thermostable antigen preparations may overcome current limitations in HPV vaccine delivery as well as being widely applicable to other antigens.

Murine polyomavirus DNA transitions through spatially distinct nuclear replication subdomains during infection.

The replication of small DNA viruses requires both host DNA replication and repair factors that are often recruited to subnuclear domains termed viral replication centers (VRCs). Aside from serving as a spatial focus for viral replication, little is known about these dynamic areas in the nucleus. We investigated the organization and function of VRCs during murine polyomavirus (MuPyV) infection using 3D structured illumination microscopy (3D-SIM). We localized MuPyV replication center components, such as the viral large T-antigen (LT) and the cellular replication protein A (RPA), to spatially distinct subdomains within VRCs. We found that viral DNA (vDNA) trafficked sequentially through these subdomains post-synthesis, suggesting their distinct functional roles in vDNA processing. Additionally, we observed disruption of VRC organization and vDNA trafficking during mutant MuPyV infections or inhibition of DNA synthesis. These results reveal a dynamic organization of VRC components that coordinates virus replication.

Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry.

The human papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are posttranslationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and bovine papillomavirus 1 (BPV-1) L2 proteins. The phospho-modification of L2 and its presence in HPV pseudovirions (PsVs) were confirmed using anti-phospho-L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiencies of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry.IMPORTANCE The papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However, nothing is known about how potential posttranslational modifications may affect different aspects of capsid assembly or infectious entry. In this study, we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the papillomavirus L2 proteins, a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway.

Viral replication centers and the DNA damage response in JC virus-infected cells.

JCV is a human polyomavirus (PyV) that establishes a persistent infection in its host. Current immunomodulatory therapies, such as Natalizumab for multiple sclerosis, can result in JCV reactivation, leading to the debilitating brain disease progressive multifocal leukoencephalopathy (PML). JCV is among the viruses that recruit and modulate the host DNA damage response (DDR) to replicate its genome. We have identified host proteins recruited to the nuclear sites of JC viral DNA (vDNA) replication using three cell types susceptible to infection in vitro. Using confocal microscopy, we found that JCV recruited a similar repertoire of host DDR proteins to these replication sites previously observed for other PyVs. Electron tomography of JCV "virus factories" showed structural features like those described for murine PyV. These results confirm and extend previous observations for PyVs to JCV emphasizing a similar replication strategy among members of this virus family.

Murine Polyomavirus Cell Surface Receptors Activate Distinct Signaling Pathways Required for Infection.

Virus binding to the cell surface triggers an array of host responses, including activation of specific signaling pathways that facilitate steps in virus entry. Using mouse polyomavirus (MuPyV), we identified host signaling pathways activated upon virus binding to mouse embryonic fibroblasts (MEFs). Pathways activated by MuPyV included the phosphatidylinositol 3-kinase (PI3K), FAK/SRC, and mitogen-activated protein kinase (MAPK) pathways. Gangliosides and α4-integrin are required receptors for MuPyV infection. MuPyV binding to both gangliosides and the α4-integrin receptors was required for activation of the PI3K pathway; however, either receptor interaction alone was sufficient for activation of the MAPK pathway. Using small-molecule inhibitors, we confirmed that the PI3K and FAK/SRC pathways were required for MuPyV infection, while the MAPK pathway was dispensable. Mechanistically, the PI3K pathway was required for MuPyV endocytosis, while the FAK/SRC pathway enabled trafficking of MuPyV along microtubules. Thus, MuPyV interactions with specific cell surface receptors facilitate activation of signaling pathways required for virus entry and trafficking. Understanding how different viruses manipulate cell signaling pathways through interactions with host receptors could lead to the identification of new therapeutic targets for viral infection. IMPORTANCE: Virus binding to cell surface receptors initiates outside-in signaling that leads to virus endocytosis and subsequent virus trafficking. How different viruses manipulate cell signaling through interactions with host receptors remains unclear, and elucidation of the specific receptors and signaling pathways required for virus infection may lead to new therapeutic targets. In this study, we determined that gangliosides and α4-integrin mediate mouse polyomavirus (MuPyV) activation of host signaling pathways. Of these pathways, the PI3K and FAK/SRC pathways were required for MuPyV infection. Both the PI3K and FAK/SRC pathways have been implicated in human diseases, such as heart disease and cancer, and inhibitors directed against these pathways are currently being investigated as therapies. It is possible that these pathways play a role in human PyV infections and could be targeted to inhibit PyV infection in immunosuppressed patients.

WU and KI polyomavirus infections in Filipino children with lower respiratory tract disease.

BACKGROUND: WU and KI are human polyomaviruses initially detected in the respiratory tract, whose clinical significance remains uncertain. OBJECTIVES: To determine the epidemiology, viral load and clinical characteristics of WU and KI polyomaviruses. STUDY DESIGN: We tested respiratory specimens collected during a randomized, placebo-controlled pneumococcal conjugate vaccine trial and related epidemiological study in the Philippines. We analyzed 1077 nasal washes from patients aged 6 weeks to 5 years who developed lower respiratory tract illness using quantitative real-time PCR for WU and KI. We collected data regarding presenting symptoms, signs, radiographic findings, laboratory data and coinfection. RESULTS: The prevalence and co-infection rates for WU were 5.3% and 74% respectively and 4.2% and 84% respectively for KI. Higher KI viral loads were observed in patients with severe or very severe pneumonia, those presenting with chest indrawing, hypoxia without wheeze, convulsions, and with KI monoinfection compared with co-infection. There was no significant association between viral load and clinical presentation for WU. CONCLUSIONS: These findings suggest a potential pathogenic role for KI, and that there is an association between KI viral load and illness severity.

Activation of DNA damage repair pathways by murine polyomavirus.

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble.

Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

Ganglioside and Non-ganglioside Mediated Host Responses to the Mouse Polyomavirus.

Gangliosides serve as receptors for internalization and infection by members of the polyomavirus family. Specificity is determined by recognition of carbohydrate moieties on the ganglioside by the major viral capsid protein VP1. For the mouse polyomavirus (MuPyV), gangliosides with terminal sialic acids in specific linkages are essential. Although many biochemical and cell culture experiments have implicated gangliosides as MuPyV receptions, the role of gangliosides in the MuPyV-infected mouse has not been investigated. Here we report results of studies using ganglioside-deficient mice and derived cell lines. Knockout mice lacking complex gangliosides were completely resistant to the cytolytic and pathogenic effects of the virus. Embryo fibroblasts from these mice were likewise resistant to infection, and supplementation with specific gangliosides restored infectibility. Although lacking receptors for viral infection, cells from ganglioside-deficient mice retained the ability to respond to the virus. Ganglioside-deficient fibroblasts responded rapidly to virus exposure with a transient induction of c-fos as an early manifestation of a mitogenic response. Additionally, splenocytes from ganglioside-deficient mice responded to MuPyV by secretion of IL-12, previously recognized as a key mediator of the innate immune response. Thus, while gangliosides are essential for infection in the animal, gangliosides are not required for mitogenic responses and innate immune responses to the virus.

Development of a highly thermostable, adjuvanted human papillomavirus vaccine.

A major impediment to economical, worldwide vaccine distribution is the requirement for a "cold chain" to preserve antigenicity. We addressed this problem using a model human papillomavirus (HPV) vaccine stabilized by immobilizing HPV16 L1 capsomeres, i.e., pentameric subunits of the virus capsid, within organic glasses formed by lyophilization. Lyophilized glass and liquid vaccine formulations were incubated at 50°C for 12weeks, and then analyzed for retention of capsomere conformational integrity and the ability to elicit neutralizing antibody responses after immunization of BALB/c mice. Capsomeres in glassy-state vaccines retained tertiary and quaternary structure, and critical conformational epitopes. Moreover, glassy formulations adjuvanted with aluminum hydroxide or aluminum hydroxide and glycopyranoside lipid A were not only as immunogenic as the commercially available HPV vaccine Cervarix®, but also retained complete neutralizing immunogenicity after high-temperature storage. The thermal stability of such adjuvanted vaccine powder preparations may thus eliminate the need for the cold chain.

Malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors.

Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. The MWPyV small T antigen (ST) binds protein phosphatase 2A (PP2A), and the large T antigen (LT) binds pRb, p107, p130, and p53. However, the MWPyV LT was less stable than the simian virus 40 (SV40) LT and was unable to promote the growth of normal cells. This report confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.

Common exposure to STL polyomavirus during childhood.

STL polyomavirus (STLPyV) was recently identified in human specimens. To determine seropositivity for STLPyV, we developed an ELISA and screened patient samples from 2 US cities (Denver, Colorado [500]; St. Louis, Missouri [419]). Overall seropositivity was 68%-70%. The age-stratified data suggest that STLPyV infection is widespread and commonly acquired during childhood.