Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses.

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.

Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.

UNLABELLED: Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE: Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses.

Uukuniemi Phlebovirus assembly and secretion leave a functional imprint on the virion glycome.

Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome.

Genome-wide small interfering RNA screens reveal VAMP3 as a novel host factor required for Uukuniemi virus late penetration.

UNLABELLED: The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3(+) endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3(+) virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE: Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.

Oligomerization of Uukuniemi virus nucleocapsid protein.

BACKGROUND: Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail. RESULTS: A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact alpha-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two alpha-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays. CONCLUSIONS: UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.

Entry of bunyaviruses into mammalian cells.

The Bunyaviridae constitute a large family of enveloped animal viruses, many members of which cause serious diseases. However, early bunyavirus-host cell interactions and entry mechanisms remain largely uncharacterized. Investigating Uukuniemi virus, a bunyavirus of the genus Phlebovirus, we found that virus attachment to the cell surface was specific but inefficient, with 25% of bound viruses being endocytosed within 10 min, mainly via noncoated vesicles. The viruses entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomes. Acid-activated penetration, occurring 20-40 min after internalization, required maturation of early to late endosomes. The pH threshold for viral membrane fusion was 5.4, and entry was sensitive to temperatures below 25 degrees C. Together, our results indicate that Uukuniemi virus penetrates host cells by acid-activated membrane fusion from late endosomal compartments. This study also highlights the importance of the degradative branch of the endocytic pathway in facilitating entry of late-penetrating viruses.

The cytoplasmic tails of Uukuniemi Virus (Bunyaviridae) G(N) and G(C) glycoproteins are important for intracellular targeting and the budding of virus-like particles.

Functional motifs within the cytoplasmic tails of the two glycoproteins G(N) and G(C) of Uukuniemi virus (UUK) (Bunyaviridae family) were identified with the help of our recently developed virus-like particle (VLP) system for UUK virus (A. K. Overby, V. Popov, E. P. Neve, and R. F. Pettersson, J. Virol. 80:10428-10435, 2006). We previously reported that information necessary for the packaging of ribonucleoproteins into VLPs is located within the G(N) cytoplasmic tail (A. K. Overby, R. F. Pettersson, and E. P. Neve, J. Virol. 81:3198-3205, 2007). The G(N) glycoprotein cytoplasmic tail specifically interacts with the ribonucleoproteins and is critical for genome packaging. In addition, two other regions in the G(N) cytoplasmic tail, encompassing residues 21 to 25 and 46 to 50, were shown to be important for particle generation and release. By the introduction of point mutations within these two regions, we demonstrate that leucines at positions 23 and 24 are crucial for the initiation of VLP budding, while leucine 46, glutamate 47, and leucine 50 are important for efficient exit from the endoplasmic reticulum and subsequent transport to the Golgi complex. We found that budding and particle generation are highly dependent on the intracellular localization of both glycoproteins. The short cytoplasmic tail of UUK G(C) contains a lysine at position -3 from the C terminus that is highly conserved among members of the Phlebovirus, Hantavirus, and Orthobunyavirus genera. Mutating this single amino acid residue in G(C) resulted in the mislocalization of not only G(C) but also G(N) to the plasma membrane, and VLP generation was compromised in cells expressing this mutant. Together, these results demonstrate that the cytoplasmic tails of both G(N) and G(C) contain specific information necessary for efficient virus particle generation.

Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding.

In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant. The particles are able to infect new cells, and reporter gene expression can be monitored if the trans-acting viral proteins (RNA polymerase and nucleoprotein) are also expressed in these cells. No minigenome transfer occurred in the absence of glycoproteins, demonstrating that the glycoproteins are absolutely required for the generation of infectious particles. Moreover, expression of glycoproteins alone was sufficient to produce and release VLPs. We show that the ribonucleoproteins (RNPs) are incorporated into VLPs but are not required for the generation of particles. Morphological analysis of the particles by electron microscopy revealed that VLPs, either with or without minigenomes, display a surface morphology indistinguishable from that of the authentic UUK virus and that they bud into Golgi vesicles in the same way as UUK virus does. This infectious VLP system will be very useful for studying the bunyaviral structural components required for budding and packaging of RNPs and receptor binding and may also be useful for the development of new vaccines for the human pathogens from this family.

Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex.

Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.