Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription, an indispensable step in natural infection. Here we describe a ZEBOV iVLP system which overcomes this limitation and show that VP24 is essential for the formation of a functional ribonucleoprotein complex.Ebola virus (EBOV) causes severe hemorrhagic fever in humans, for which treatment is currently unavailable (6, 8). To study particle morphogenesis, genome packaging, and virion entry into target cells under biosafety level 2 conditions, an infectious virus-like particle (iVLP) system for Zaire ebolavirus (ZEBOV) was established (20). This system extends the original minigenome systems (4, 7, 14) by including VP24, VP40, and GP to produce iVLPs which resemble wild-type (WT) virions but contain minigenomes instead of full-length viral genomes. These iVLPs can deliver the minigenomes to target cells, where they are transcribed and replicated in the presence of helper ribonucleoprotein (RNP) components (the nucleoprotein [NP], VP35, VP30, and L components). Similar systems are available for other negative-sense RNA viruses, but RNP components must always be provided to target cells in trans, via either helper virus infection or expression plasmid transfection (12,(15)(16)(17)(19)(20)(21). Therefore, it is impossible to determine whether iVLPs contain fully functional RNP complexes or whether they contain minigenomes but are unable to facilitate initial transcription, as is necessary in virus infection. EBOV VP24 has been described as a minor matrix protein, but its contribution to budding is controversial (10, 13). Electron microscopic studies imply a role in the formation of nucleocapsid-like structures (11), while another study suggests that VP24 is unnecessary for the packaging and delivery of minigenomes by iVLPs (20). To better understand the contribution of VP24 to morphogenesis, we established an iVLP system with naïve target cells to allow assessment of iVLP-associated RNP complex function. As a first step, an iVLP system based on that previously described (20) was established in our laboratory (Fig. 1A). 293T cells (p0) were transfected using FuGENE (Roche) with plasmids encoding each ZEBOV structural protein (125 ng pCAGGS-NP, 125 ng pCAGGS-VP35, 75 ng pCAGGS-VP30, 1,000 ng pCAGGS-L, 250 ng pCAGGS-GP, 250 ng pCAGGS-VP40, and 60 ng pCAGGS-VP24) and a T7-driven minigenome encoding a Renilla luciferase reporter (250 ng), which is detectable in minute amounts (23). Cell supernatant containing released iVLPs was harvested 3 days posttransfection, cleared of cellular debris, and used to infect target 293T cells (p1) previ...

The Marburg virus (MARV) envelope consists of a lipid membrane and two major proteins, the matrix protein VP40 and the glycoprotein GP. Both proteins use different intracellular transport pathways: GP utilizes the exocytotic pathway, while VP40 is transported through the retrograde late endosomal pathway. It is currently unknown where the proteins combine to form the viral envelope. In the present study, we identified the intracellular site where the two major envelope proteins of MARV come together as peripheral multivesicular bodies (MVBs). Upon coexpression with VP40, GP is redistributed from the trans-Golgi network into the VP40-containing MVBs. Ultrastructural analysis of MVBs suggested that they provide the platform for the formation of membrane structures that bud as virus-like particles from the cell surface. The virus-like particles contain both VP40 and GP. Single expression of GP also resulted in the release of particles, which are round or pleomorphic. Single expression of VP40 led to the release of filamentous structures that closely resemble viral particles and contain traces of endosomal marker proteins. This finding indicated a central role of VP40 in the formation of the filamentous structure of MARV particles, which is similar to the role of the related Ebola virusVP40. In MARV-infected cells, VP40 and GP are colocalized in peripheral MVBs as well. Moreover, intracellular budding of progeny virions into MVBs was frequently detected. Taken together, these results demonstrate an intracellular intersection between GP and VP40 pathways and suggest a crucial role of the late endosomal compartment for the formation of the viral envelope.

. 76:1825-1838, 2002). The present study revealed that VP40 is bound to cellular membranes rapidly after synthesis. Time course studies were performed to trace the distribution of VP40 during the course of expression. First, VP40 was homogenously distributed throughout the cytoplasm, although the majority of protein (70%) was already membrane associated. Next, VP40 accumulated in MVBs and in tubular protrusions emerging from MVBs. Finally, VP40 appeared in a patch-like pattern beneath the plasma membrane. These morphological results were supported by iodixanol density gradient analyses. The majority of VP40-positive membranes were first detected comigrating with small vesicles. VP40 was then shifted to fractions containing endosomal marker proteins, and later, to fractions containing plasma membrane marker proteins. Blocking of protein synthesis by use of cycloheximide at the time when VP40 was mainly associated with the small vesicles did not prevent the redistribution of VP40 to the late endosomes and further to the plasma membrane. The inhibition of intracellular vesicular trafficking by monensin significantly reduced the appearance of VP40 at the plasma membrane. In conclusion, we suggest that the transport of the Marburg virus matrix protein VP40 involves its accumulation in MVBs followed by the redistribution of VP40-enriched membrane clusters to the plasma membrane.

The highly pathogenic enveloped Marburg virus (MARV) is composed of seven structural proteins and the nonsegmented negative-sense viral RNA genome. Four proteins (NP, VP35, VP30, and L) make up the helical nucleocapsid, which is surrounded by a matrix that is composed of the viral proteins VP40 and VP24. VP40 is functionally homologous to the matrix proteins of other nonsegmented negative-strand RNA viruses. As yet, the function of VP24 remains elusive. In the present study we found that VP24 colocalized with inclusions in MARV-infected cells that contain preformed nucleocapsids and with nucleocapsids outside the inclusions. Coexpression studies revealed that VP24 is recruited into the inclusions by the presence of NP. Furthermore, VP24 displayed membrane-binding properties and was recruited into filamentous virus-like particles (VLPs) that are induced by VP40. The incorporation of VP24 altered neither the morphology of VLPs nor the budding efficiency of VLPs. When VP24 was silenced in MARV-infected cells by small interfering RNA technology, the release of viral particles was significantly reduced while viral transcription and replication were unimpaired. Our data support the idea that VP24 is essential for a process that takes place after replication and transcription and before budding of virus progeny. It is presumed that VP24 is necessary for the formation of transport-competent nucleocapsids and/or the interaction between the nucleocapsids and the budding sites at the plasma membrane.