Methylation of viral mRNA cap structures by PCIF1 attenuates the antiviral activity of interferon-ß.

Interferons induce cell-intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. Using vesicular stomatitis virus (VSV), we report that the host cell N6-adenosine messenger RNA (mRNA) cap methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m7Gpppm6Am and define the substrate requirements for this modification. Functional assays revealed that the PCIF1-dependent modification of VSV mRNA cap structures is inert with regard to mRNA stability, translation, and viral infectivity but attenuates the antiviral effects of the treatment of cells with interferon-ß. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-ß treatment. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m7Gpppm6Am This work identifies a function of PCIF1 and cap-proximal m6Am in attenuation of the host response to VSV infection that likely extends to other viruses.

Untargeted Lipidomics of Vesicular Stomatitis Virus-Infected Cells and Viral Particles.

The viral lifecycle is critically dependent upon host lipids. Enveloped viral entry requires fusion between viral and cellular membranes. Once an infection has occurred, viruses may rely on host lipids for replication and egress. Upon exit, enveloped viruses derive their lipid bilayer from host membranes during the budding process. Furthermore, host lipid metabolism and signaling are often hijacked to facilitate viral replication. We employed an untargeted HILIC-IM-MS lipidomics approach and identified host lipid species that were significantly altered during vesicular stomatitis virus (VSV) infection. Many glycerophospholipid and sphingolipid species were modified, and ontological enrichment analysis suggested that the alterations to the lipid profile change host membrane properties. Lysophosphatidylcholine (LPC), which can contribute to membrane curvature and serve as a signaling molecule, was depleted during infection, while several ceramide sphingolipids were augmented during infection. Ceramide and sphingomyelin lipids were also enriched in viral particles, indicating that sphingolipid metabolism is important during VSV infection.

Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein.

Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

Negative-Strand RNA Virus L Proteins: One Machine, Many Activities.

Structures of L proteins from La Crosse orthobunyavirus and vesicular stomatitis virus reveal insights into RNA synthesis and distinctive mRNA capping mechanisms of segmented and non-segmented negative-sense single-strand RNA viruses.

Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy.

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

Switchable aptamers for biosensing and bioseparation of viruses (SwAps-V).

There is a widespread interest in the development of aptamer-based affinity chromatographic methods for purification of biomolecules. Regardless of the many advantages exhibited by aptamers when compared to other recognition elements, the lack of an efficient regeneration technique that can be generalized to all targets has encumbered further integration of aptamers into affinity-based purification methods. Here we offer switchable aptamers (SwAps) that have been developed to solve this problem and move aptamer-based chromatography forward. SwAps are controlled-affinity aptamers, which have been employed here to purify vesicular stomatitis virus (VSV) as a model case, however this technique can be extended to all biologically significant molecules. VSV is one oncolytic virus out of an arsenal of potential candidates shown to provide selective destruction of cancer cells both in vitro and in vivo. These SwAps were developed in the presence of Ca(2+) and Mg(2+) ions where they cannot bind to their target VSV in absence of these cations. Upon addition of EDTA and EGTA, the divalent cations were sequestered from the stabilized aptameric structure causing a conformational change and subsequently release of the virus. Both flow cytometry and electrochemical impedance spectroscopy were employed to estimate the binding affinities between the selected SwAps and VSV and to determine the coefficient of switching (CoS) upon elution. Among fifteen sequenced SwAps, four have exhibited high affinity to VSV and ability to switch upon elution and thus were further integrated into streptavidin-coated magnetic beads for purification of VSV.

Cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions.

Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact ("whole") virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.

Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

pH-dependent molecular dynamics of vesicular stomatitis virus glycoprotein G.

Vesicular stomatitis virus glycoprotein G (VSV-G) belongs to a new class of viral fusion proteins (Class III). The structure of VSV-G has been solved in two different conformations and fusion is known to be triggered by low pH. To investigate Class III fusion mechanisms, molecular dynamics simulations were performed on the VSV-G prefusion structure in two different protonation states: at physiological pH (pH 7) and low pH present in the endosome (pH 5). Domain IV containing the fusion loops, which need to interact with the target membrane, exhibits the highest mobility. Energetic analyses revealed weakened interaction between Domain IV and the protein core at pH 5, which can be attributed to two pairs of structurally neighboring conserved and differentially protonated residues in the Domain IV-core interface. Energetic calculations also demonstrated that the interaction between the subunits in the core of the trimeric VSV-G is strengthened at pH 5, mainly due to newly formed interactions between the C-terminal loop of Domain II and the N-terminus of the adjacent subunit. A pair of interacting residues in this interface that is affected by differential protonation was shown to be the main effectors of this phenomenon. The results of this study thus enhance the mechanistic understanding of the effects of protonation changes in VSV-G.

Electrochemical sensing of aptamer-facilitated virus immunoshielding.

Oncolytic viruses (OVs) are promising therapeutics that selectively replicate in and kill tumor cells. However, repetitive administration of OVs provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effects. In this work, we selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus (VSV), to protect it from nAbs. A label-free electrochemical aptasensor was used to evaluate the degree of protection (DoP). The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VSV-specific aptamer. Electrochemical impedance spectroscopy was employed to quantitate VSV in the range of 800-2200 PFU and a detection limit of 600 PFU. The aptasensor was also utilized for evaluating binding affinities between VSV and aptamer pools/clones. An electrochemical displacement assay was performed in the presence of nAbs and DoP values were calculated for several VSV-aptamer pools/clones. A parallel flow cytometric analysis confirmed the electrochemical results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22, ZMYK-23, and ZMYK-28, showed the highest protective properties with dissociation constants of 17, 8, 20, and 13 nM, respectively. Another four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities to VSV without protecting it from nAbs and can be further utilized in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential to allow efficient delivery of VSV through the bloodstream without compromising the patient's immune system.

Kinetics of virus production from single cells.

The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.

An unconventional pathway of mRNA cap formation by vesiculoviruses.

mRNAs of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative strand (NNS) RNA viruses (e.g., rabies, measles, mumps, Ebola, and Borna disease viruses), possess the 5'-terminal cap structure identical to that of eukaryotic mRNAs, but the mechanism of mRNA cap formation is distinctly different from the latter. The elucidation of the unconventional capping of VSV mRNA remained elusive for three decades since the discovery of the cap structure in some viral and eukaryotic mRNAs in 1975. Only recently our biochemical studies revealed an unexpected strategy employed by vesiculoviruses (VSV and Chandipura virus, an emerging arbovirus) to generate the cap structure. This article summarizes the historical and current research that led to the discovery of the novel vesiculoviral mRNA capping reaction.

Fluorescein and radiolabeled Function-Spacer-Lipid constructs allow for simple in vitro and in vivo bioimaging of enveloped virions.

Tools that can aid in vitro and in vivo imaging and also noninvasively determine half-life and biodistribution are required to advance clinical developments. A Function-Spacer-Lipid construct (FSL) incorporating fluorescein (FSL-FLRO4) was used to label vesicular stomatitis virus (VSV), measles virus MV-NIS (MV) and influenza virus (H1N1). The ability of FSL constructs to label these virions was established directly by FACScan of FSL-FLRO4 labeled VSV and MV, and indirectly following labeled H1N1 and MV binding to a cells. FSL-FLRO4 labeling of H1N1 was shown to maintain higher infectivity of the virus when compared with direct fluorescein virus labeling. A novel tyrosine (125)I radioiodinated FSL construct was synthesized (FSL-(125)I) from FSL-tyrosine. This was used to label VSV (VSV-FSL-(125)I), which was infused into the peritoneal cavity of laboratory mice. Bioscanning showed VSV-FSL-(125)I to localize in the liver, spleen and bloodstream in contrast to the free labels FSL-(125)I or (125)I, which localized predominantly in the liver and thyroid respectively. This is a proof-of-principle novel and rapid method for modifying virions and demonstrates the potential of FSL constructs to improve in vivo imaging of virions and noninvasively observe in vivo biodistribution.

Structural and functional properties of the vesicular stomatitis virus nucleoprotein-RNA complex as revealed by proteolytic digestion.

To gain insight into the structural and functional properties of the vesicular stomatitis virus nucleocapsid-RNA complex (vN-RNA), we analyzed it by treatment with proteolytic enzymes. Chymotrypsin treatment to the vN-RNA results in complete digestion of the C-terminal 86 amino acids of the N protein. The residual chymotrypsin resistant vN-RNA complex (vDeltaN-RNA) carrying N-terminal 336 amino acids of the N protein (DeltaN) was inactive in transcription. The DeltaN protein retained its capability to protect the genomic RNA from nuclease digestion but failed to interact to the P protein. Interestingly, addition of excess amount of P protein rendered the vN-RNA complex resistant to the chymotrypsin digestion. Finally, our data revealed that the recombinant N-RNA complex purified from bacteria (bN-RNA) is resistant to chymotrypsin digestion, suggesting that the C-terminal unstructured domain (C-loop) remains inaccessible to protease digestion. Detailed comparative analyses of the vN-RNA and vDeltaN-RNA are discussed.

Structure of the dimerization domain of the rabies virus phosphoprotein.

The crystal structure of the dimerization domain of rabies virus phosphoprotein was determined. The monomer consists of two alpha-helices that make a helical hairpin held together mainly by hydrophobic interactions. The monomer has a hydrophilic and a hydrophobic face, and in the dimer two monomers pack together through their hydrophobic surfaces. This structure is very different from the dimerization domain of the vesicular stomatitis virus phosphoprotein and also from the tetramerization domain of the Sendai virus phosphoprotein, suggesting that oligomerization is conserved but not structure.

Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor, P.

The negative-strand RNA viruses (NSRVs) are unique because their nucleocapsid, not the naked RNA, is the active template for transcription and replication. The viral polymerase of nonsegmented NSRVs contains a large polymerase catalytic subunit (L) and a nonenzymatic cofactor, the phosphoprotein (P). Insight into how P delivers the polymerase complex to the nucleocapsid has long been pursued by reverse genetics and biochemical approaches. Here, we present the X-ray crystal structure of the C-terminal domain of P of vesicular stomatitis virus, a prototypic nonsegmented NSRV, bound to nucleocapsid-like particles. P binds primarily to the C-terminal lobe of 2 adjacent N proteins within the nucleocapsid. This binding mode is exclusive to the nucleocapsid, not the nucleocapsid (N) protein in other existing forms. Localization of phosphorylation sites within P and their proximity to the RNA cavity give insight into how the L protein might be oriented to access the RNA template.

Solution structure of the C-terminal nucleoprotein-RNA binding domain of the vesicular stomatitis virus phosphoprotein.

Beyond common features in their genome organization and replication mechanisms, the evolutionary relationships among viruses of the Rhabdoviridae family are difficult to decipher because of the great variability in the amino acid sequence of their proteins. The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential component of the RNA transcription and replication machinery; in particular, it contains binding sites for the RNA-dependent RNA polymerase and for the nucleoprotein. Here, we devised a new method for defining boundaries of structured domains from multiple disorder prediction algorithms, and we identified an autonomous folding C-terminal domain in VSV P (P(CTD)). We show that, like the C-terminal domain of rabies virus (RV) P, VSV P(CTD) binds to the viral nucleocapsid (nucleoprotein-RNA complex). We solved the three-dimensional structure of VSV P(CTD) by NMR spectroscopy and found that the topology of its polypeptide chain resembles that of RV P(CTD). The common part of both proteins could be superimposed with a backbone RMSD from mean atomic coordinates of 2.6 A. VSV P(CTD) has a shorter N-terminal helix (alpha(1)) than RV P(CTD); it lacks two alpha-helices (helices alpha(3) and alpha(6) of RV P), and the loop between strands beta(1) and beta(2) is longer than that in RV. Dynamical properties measured by NMR relaxation revealed the presence of fast motions (below the nanosecond timescale) in loop regions (amino acids 209-214) and slower conformational exchange in the N- and C-terminal helices. Characterization of a longer construct indicated that P(CTD) is preceded by a flexible linker. The results presented here support a modular organization of VSV P, with independent folded domains separated by flexible linkers, which is conserved among different genera of Rhabdoviridae and is similar to that proposed for the P proteins of the Paramyxoviridae.

Molecular architecture of the bipartite fusion loops of vesicular stomatitis virus glycoprotein G, a class III viral fusion protein.

The glycoprotein of vesicular stomatitis virus (VSV G) mediates fusion of the viral envelope with the host cell, with the conformational changes that mediate VSV G fusion activation occurring in a reversible, low pH-dependent manner. Based on its novel structure, VSV G has been classified as class III viral fusion protein, having a predicted bipartite fusion domain comprising residues Trp-72, Tyr-73, Tyr-116, and Ala-117 that interacts with the host cell membrane to initiate the fusion reaction. Here, we carried out a systematic mutagenesis study of the predicted VSV G fusion loops, to investigate the functional role of the fusion domain. Using assays of low pH-induced cell-cell fusion and infection studies of mutant VSV G incorporated into viral particles, we show a fundamental role for the bipartite fusion domain. We show that Trp-72 is a critical residue for VSV G-mediated membrane fusion. Trp-72 could only tolerate mutation to a phenylalanine residue, which allowed only limited fusion. Tyr-73 and Tyr-116 could be mutated to other aromatic residues without major effect but could not tolerate any other substitution. Ala-117 was a less critical residue, with only charged residues unable to allow fusion activation. These data represent a functional analysis of predicted bipartite fusion loops of VSV G, a founder member of the class III family of viral fusion proteins.

Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb.

BACKGROUND: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. METHODS: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. RESULTS: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. CONCLUSION: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.