Adeno-associated virus capsid protein expression in Escherichia coli and chemically defined capsid assembly.

Research and clinical applications of recombinant adeno-associated virus (rAAV) significantly increased in recent years alongside regulatory approvals of rAAV gene therapy products. To date, all rAAV vectors as well as AAV empty capsids are produced in eukaryotic cells. We explored a new route to generate AAV capsids with the aim to analyze capsid assembly in a chemically defined setting and pave the way for new production methods and applications based on AAV virus-like particles (VLPs). We generated these empty capsids by bacterial expression and subsequent concomitant protein refolding and VLP formation. AAV serotype 2 structural protein VP3 was expressed in Escherichia coli. VLPs formed as demonstrated by dynamic light scattering, atomic force microscopy, and ELISA. Furthermore, VLPs internalized into human HeLa cells. To extend the application range of the VLPs, we tested peptide insertions, at the genetic level, in a surface loop (amino acid position 587) or at the C-terminus of VP3 and these variants also formed VLPs. VLPs developed without assembly-activating protein (AAP), but adding purified recombinant AAP to the refolding process increased capsid yield. Our findings offer a new route to understand AAV assembly biology and open a toolbox for AAV production strategies that might enable capsid display for vaccination and matching of capsids with cargoes at large scale and low cost.

Molecular Piracy: Redirection of Bacteriophage Capsid Assembly by Mobile Genetic Elements.

Horizontal transfer of mobile genetic elements (MGEs) is a key aspect of the evolution of bacterial pathogens. Transduction by bacteriophages is especially important in this process. Bacteriophages-which assemble a machinery for efficient encapsidation and transfer of genetic material-often transfer MGEs and other chromosomal DNA in a more-or-less nonspecific low-frequency process known as generalized transduction. However, some MGEs have evolved highly specific mechanisms to take advantage of bacteriophages for their own propagation and high-frequency transfer while strongly interfering with phage production-"molecular piracy". These mechanisms include the ability to sense the presence of a phage entering lytic growth, specific recognition and packaging of MGE genomes into phage capsids, and the redirection of the phage assembly pathway to form capsids with a size more appropriate for the size of the MGE. This review focuses on the process of assembly redirection, which has evolved convergently in many different MGEs from across the bacterial universe. The diverse mechanisms that exist suggest that size redirection is an evolutionarily advantageous strategy for many MGEs.

Evolutionary study of potentially zoonotic hepatitis E virus genotype 3 from swine in Northeast Brazil.

Hepatitis E virus (HEV), an emerging virus associated with acute hepatic disease, leads to thousands of deaths worldwide. HEV has already been reported in Brazil; however, there is a lack of epidemiological and molecular information on the genetic variability, taxonomy, and evolution of HEV. It is thus unclear whether hepatitis E is a neglected disease in Brazil or it has low relevance for public health in this country. Here, for the first time, we report the presence of HEV in Northeast Brazil. A total of 119 swine faecal samples were screened for the presence of HEV RNA using real-time polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR; among these, two samples were identified as positive. Molecular evolution analyses based on capsid sequences revealed that the samples had close proximities to HEV sequences belonging to genotype 3 and were genetically related to subtype 3f isolated in humans. Parsimony ancestral states analysis indicated gene flow events from HEV cross-species infection, suggesting an important role of pig hosts in viral spillover. HEV's ability for zoonotic transmission by inter-species host switching as well as its possible adaptation to new animal species remain important issues for human health.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element.

Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.

Residues on Adeno-associated Virus Capsid Lumen Dictate Interactions and Compatibility with the Assembly-Activating Protein.

The adeno-associated virus (AAV) serves as a broadly used vector system for in vivo gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly.IMPORTANCE Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.

Evolutionary study of potentially zoonotic hepatitis E virus genotype 3 from swine in Northeast Brazil

Hepatitis E virus (HEV), an emerging virus associated with acute hepatic disease, leads to thousands of deaths worldwide. HEV has already been reported in Brazil; however, there is a lack of epidemiological and molecular information on the genetic variability, taxonomy, and evolution of HEV. It is thus unclear whether hepatitis E is a neglected disease in Brazil or it has low relevance for public health in this country. Here, for the first time, we report the presence of HEV in Northeast Brazil. A total of 119 swine faecal samples were screened for the presence of HEV RNA using real-time polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR; among these, two samples were identified as positive. Molecular evolution analyses based on capsid sequences revealed that the samples had close proximities to HEV sequences belonging to genotype 3 and were genetically related to subtype 3f isolated in humans. Parsimony ancestral states analysis indicated gene flow events from HEV cross-species infection, suggesting an important role of pig hosts in viral spillover. HEV's ability for zoonotic transmission by inter-species host switching as well as its possible adaptation to new animal species remain important issues for human health.

Non-human papillomaviruses for gene delivery in vitro and in vivo.

Papillomavirus capsids are known to have the ability to package DNA plasmids and deliver them both in vitro and in vivo. Of all known papillomavirus types, human papillomaviruses (HPVs) are by far the most intensely studied. Although HPVs work well as gene transfer vectors, their use is limited as most individuals are exposed to this virus either through a HPV vaccination or natural infection. To circumvent these constraints, we produced pseudovirions (PsVs) of ten non-human papillomavirus types and tested their transduction efficiencies in vitro. PsVs based on Macaca fascicularis papillomavirus-11 and Puma concolor papillomavirus-1 were further tested in vivo. Intramuscular transduction by PsVs led to months-long expression of a reporter plasmid, indicating that PsVs have potential as gene delivery vectors.

Encapsulation of Negatively Charged Cargo in MS2 Viral Capsids.

Encapsulation into virus-like particles is an efficient way of loading cargo of interest for delivery applications. Here, we describe the encapsulation of proteins with tags comprising anionic amino acids or DNA and gold nanoparticles with negative surface charges inside MS2 bacteriophage capsids to obtain homogeneous nanoparticles with a diameter of 27 nm.

Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain.

Intravenous administration of adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV9 containing seven amino acid insertions, results in a greater permeability of the blood brain barrier (BBB) than standard AAV9 in mice, leading to highly efficient and global transduction of the central nervous system (CNS). The present study aimed to examine whether the enhanced BBB penetrance of AAV-PHP.B observed in mice also occurs in non-human primates. Thus, a young adult (age, 1.6 years) and an old adult (age, 7.2 years) marmoset received an intravenous injection of AAV-PHP.B expressing enhanced green fluorescent protein (EGFP) under the control of the constitutive CBh promoter (a hybrid of cytomegalovirus early enhancer and chicken ß-actin promoter). Age-matched control marmosets were treated with standard AAV9-capsid vectors. The animals were sacrificed 6 weeks after the viral injection. Based on the results, only limited transduction of neurons (0-2%) and astrocytes (0.1-2.5%) was observed in both AAV-PHP.B- and AAV9-treated marmosets. One noticeable difference between AAV-PHP.B and AAV9 was the marked transduction of the peripheral dorsal root ganglia neurons. Indeed, the soma and axons in the projection from the spinal cord to the nucleus cuneatus in the medulla oblongata were strongly labeled with EGFP by AAV-PHP.B. Thus, except for the peripheral dorsal root ganglia neurons, the AAV-PHP.B transduction efficiency in the CNS of marmosets was comparable to that of AAV9 vectors.

Reactivation of Herpes Zoster Keratitis With Corneal Perforation After Zoster Vaccination.

PURPOSE: We present a case of reactivated herpes zoster keratouveitis of 6 years duration with corneal perforation requiring penetrating keratoplasty shortly after inoculation with herpes zoster vaccine (Zostavax, Merck, Quebec, Canada). METHODS: Retrospective case report. RESULTS: A 67-year-old woman with a 5-year history of recurrent unilateral herpes zoster keratouveitis in her right eye presented with another recurrence 2 weeks after Zostavax vaccination. Three months later, she developed descemetocele and 2 months afterward, corneal perforation, which was managed by penetrating keratoplasty. Immunohistopathological examination disclosed positive staining for varicella zoster virus in most of the keratocytes adjacent to the descemetocele and perforation, most vividly in the deeper two-thirds of the stroma where the keratocytes were most dense, but not in corneal epithelium or endothelium. Electron microscopic examination showed universally severely degenerated corneal keratocytes in the corneal stroma adjacent to the perforation with variable numbers of herpes virus capsids present in half of these cells. Only a rare normal-appearing keratocyte was identified in the more peripheral corneal stroma. CONCLUSIONS: We present a case of reactivation of herpes keratouveitis shortly after vaccination with Zostavax in a patient with previous herpes zoster ophthalmicus. We demonstrate, for the first time, ultrastructural evidence consistent with inactive virus capsids in diffusely degenerated keratocytes in the extracted corneal tissue.

A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles.

The rodent protoparvovirus H-1PV, with its oncolytic and oncosuppressive properties, is a promising anticancer agent currently under testing in clinical trials. This explains the current demand for a scalable, good manufacturing practice-compatible virus purification process yielding high-grade pure infectious particles and overcoming the limitations of the current system based on density gradient centrifugation. We describe here a scalable process offering high purity and recovery. Taking advantage of the isoelectric point difference between full and empty particles, it eliminates most empty particles. Full particles have a significantly higher cationic charge than empty ones, with an isoelectric point of 5.8-6.2 versus 6.3 (as determined by isoelectric focusing and chromatofocusing). Thanks to this difference, infectious full particles can be separated from empty particles and most protein impurities by Convective interaction media® diethylaminoethyl (DEAE) anion exchange chromatography: applying unpurified H-1PV to the column in 0.15 M NaCl leaves, the former on the column and the latter in the flow through. The full particles are then recovered by elution with 0.25 M NaCl. The whole large-scale purification process involves filtration, single-step DEAE anion exchange chromatography, buffer exchange by cross-flow filtration, and final formulation in Visipaque/Ringer solution. It results in 98% contaminating protein removal and 96% empty particle elimination. The final infectious particle concentration reaches 3.5E10 plaque forming units (PFU)/ml, with a specific activity of 6.8E11 PFU/mg protein. Overall recovery is over 40%. The newly established method is suitable for use in commercial production.

Modeling global changes induced by local perturbations to the HIV-1 capsid.

The HIV-1 capsid is a conical protein shell made up of hexamers and pentamers of the capsid protein. The capsid houses the viral genome and replication machinery, and its opening, or uncoating, within the host cell marks a critical step in the HIV-1 lifecycle. Binding of host factors such as TRIM5α and cyclophilin A (CypA) can alter the capsid's stability, accelerating or delaying the onset of uncoating and disrupting infectivity. We employ coarse-grained computational modeling to investigate the effects of point mutations and host factor binding on HIV-1 capsid stability. We find that the largest fluctuations occur in the low-curvature regions of the capsid, and that its structural dynamics are affected by perturbations at the inter-hexamer interfaces and near the CypA binding loop, suggesting roles for these features in capsid stability. Our models show that linking capsid proteins across hexamers attenuates vibration in the low-curvature regions of the capsid, but that linking within hexamers does not. These results indicate a possible mechanism through which CypA binding alters capsid stability and highlight the utility of coarse-grained network modeling for understanding capsid mechanics.

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids.

Evaluation of an Optimized Injection System for Retinal Gene Therapy in Human Patients.

Many retinal gene therapy clinical trials require subretinal injections of small volumes of adeno-associated viral (AAV) vector solutions in patients with retinal dystrophies, using equipment not specifically designed for this purpose. We therefore evaluated an optimized injection system in order to identify variables that might influence the rate of injection and final dose of vector delivered. An optimized injection system was assembled with a 41G polytetrafluoroethylene tip for retinal gene therapy. Flow rate was recorded at relevant infusion pressures (2-22 psi [14-152 kPa]), different target pressures (0.02-30 mm Hg [0.003-4 kPa]) and temperatures (18°C vs. 36°C) using a semiautomated Accurus(®) Surgical System. Retention of AAV2/8 and AAV2/8(Y733F) vector was quantified after simulating loading/injection with or without 0.001% Pluronic(®) F-68 (PF-68). The optimized injection system provided a linear flow rate (µl/s)-to-infusion pressure (psi) relationship (y = 0.62x; r(2) = 0.99), independent of temperature and pressure changes relevant for intraocular surgery (18-36°C, 0.02-30 mm Hg). Differences in length of 41G polytetrafluoroethylene tips caused significant variation in flow rate (p < 0.001). Use of PF-68 significantly (p < 0.001) reduced loss of vector genomes in the injection system by 55% (AAV2/8) and 52% (AAV2/8(Y733F)). A customized subretinal injection system assembled using equipment currently available in the operating room can deliver a controlled volume of vector at a fixed rate across a range of possible clinical parameters encountered in vitreoretinal surgery. The inclusion of 0.001% PF-68 had a significant effect on the final dose of vector genomes delivered. The described technique is currently used successfully in a clinical trial.

Genetic Diversity in the Major Capsid L1 Protein of HPV-16 and HPV-18 in the Netherlands.

OBJECTIVES: Intratypic molecular variants of human papillomavirus (HPV) type-16 and -18 exist. In the Netherlands, a bivalent vaccine, composed of recombinant L1 proteins from HPV-16 and -18, is used to prevent cervical cancer since 2009. Long-term vaccination could lead to changes in HPV-16 and -18 virus population, thereby hampering vaccination strategies. We determined the genetic diversity of the L1 gene in HPV-16 and -18 viral strains circulating in the Netherlands at the start of vaccination in order to understand the baseline genetic diversity in the Dutch population. METHODS: DNA sequences of the L1 gene were determined in HPV-16 (n = 241) and HPV-18 (n = 108) positive anogenital samples collected in 2009 and 2011 among Dutch 16- to 24-year old female and male attendees of the sexually transmitted infection (STI) clinics. Phylogenetic analysis was performed and sequences were compared to reference sequences HPV-16 (AF536179) and HPV-18 (X05015) using BioNumerics 7.1. RESULTS: For HPV-16, ninety-five single nucleotide polymorphism (SNPs) were identified, twenty-seven (28%) were non-synonymous variations. For HPV-18, seventy-one SNPs were identified, twenty-nine (41%) were non-synonymous. The majority of the non-silent variations were located in sequences encoding alpha helix, beta sheet or surface loops, in particular in the immunodominant FG loop, and may influence the protein secondary structure and immune recognition. CONCLUSIONS: This study provides unique pre-vaccination/baseline data on the genetic L1 diversity of HPV-16 and -18 viruses circulating in the Netherlands among adolescents and young adults.

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly.

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/ß1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.

Sequence analysis of the capsid gene during a genotype II.4 dominated norovirus season in one university hospital: identification of possible transmission routes.

Norovirus (NoV) is a leading cause of gastroenteritis and genotype II.4 (GII.4) is responsible for the majority of nosocomial NoV infections. Our objective was to examine whether sequencing of the capsid gene might be a useful tool for the hospital outbreak investigation to define possible transmission routes. All NoV positive samples submitted from one university hospital during the 2007/8 season were selected. Genotyping of selected samples by partial polymerase gene sequencing had shown that the majority belonged to the GII.4 variant Den Haag 2006b and had identical polymerase sequences. Sequences of the capsid gene (1412 nucleotides) were obtained from the first available sample from 55 patients. From six immunocompromised patients with persistent infections a second sample was also included. As a control for a point-source outbreak, five samples from a foodborne outbreak caused by the same GII.4 variant were analyzed. Forty-seven of the inpatients (85%) were infected with the GII.4 variant Den Haag 2006b. Phylogenetic analysis of the Den Haag 2006b sequences identified four distinct outbreaks in different departments and a fifth outbreak with possible inter-department spread. In addition, a more heterogeneous cluster with evidence of repeated introductions from the community, but also possible inter-department spread was observed. In all six patients with paired sequences, evidence for in vivo evolution of the virus was found. Capsid gene sequencing showed substantial sequence variation among NoV GII.4 variant Den Haag 2006b strains from one single institution during a nine months' period. This method proved useful to understand the local epidemiology and, when used promptly, has the potential to make infection control measures more targeted.

Structural insight into HIV-1 restriction by MxB.

The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended antiparallel dimer and dimerization, but not higher-ordered oligomerization, is critical for restriction. Although MxB is structurally similar to MxA, the orientation of individual domains differs between MxA and MxB, and their antiviral functions rely on separate determinants, indicating distinct mechanisms for virus inhibition. Additionally, MxB directly binds the HIV-1 capsid, and this interaction depends on dimerization and the N terminus of MxB as well as the assembled capsid lattice. These insights establish a framework for understanding the mechanism by which MxB restricts HIV-1.

Maturation and vesicle-mediated egress of primate gammaherpesvirus rhesus monkey rhadinovirus require inner tegument protein ORF52.

UNLABELLED: The tegument layer of herpesviruses comprises a collection of proteins that is unique to each viral species. In rhesus monkey rhadinovirus (RRV), a close relative of the human oncogenic pathogen Kaposi's sarcoma-associated herpesvirus, ORF52 is a highly abundant tegument protein tightly associated with the capsid. We now report that ORF52 knockdown during RRV infection of rhesus fibroblasts led to a greater than 300-fold reduction in the viral titer by 48 h but had little effect on the number of released particles and caused only modest reductions in the levels of intracellular viral genomic DNA and no appreciable change in viral DNA packaging into capsids. These data suggested that the lack of ORF52 resulted in the production and release of defective particles. In support of this interpretation, transmission electron microscopy (TEM) revealed that without ORF52, capsid-like particles accumulated in the cytoplasm and were unable to enter egress vesicles, where final tegumentation and envelopment normally occur. TEM also demonstrated defective particles in the medium that closely resembled the accumulating intracellular particles, having neither a full tegument nor an envelope. The disruption in tegument formation from ORF52 suppression, therefore, prevented the incorporation of ORF45, restricting its subcellular localization to the nucleus and appearing, by confocal microscopy, to inhibit particle transport toward the periphery. Ectopic expression of small interfering RNA (siRNA)-resistant ORF52 was able to partially rescue all of these phenotypic changes. In sum, our results indicate that efficient egress of maturing virions and, in agreement with studies on murine gammaherpesvirus 68 (MHV-68), complete tegumentation and secondary envelopment are dependent on intact ORF52. IMPORTANCE: The tegument, or middle layer, of herpesviruses comprises both viral and cellular proteins that play key roles in the viral life cycle. A subset of these proteins is present only within members of one of the three subfamilies (alphaherpesviruses, betaherpesviruses, or gammaherpesviruses) of Herpesviridae. In this report, we show that the gammaherpesvirus-specific tegument protein ORF52 is critical for maturation of RRV, the closest relative of Kaposi's sarcoma-associated herpesvirus (KSHV) (a human cancer-causing pathogen) that has undergone this type of analysis. Without ORF52, the nascent subviral particles are essentially stuck in maturation limbo, unable to acquire the tegument or outer (envelope) layers. This greatly attenuates infectivity. Our data, together with earlier work on a murine homolog, as well as a more distantly related human homolog, provide a more complete understanding of how early protein interactions involving virus-encoded tegument proteins are critical for virus assembly and are also, therefore, potentially attractive therapeutic targets.