In-depth analysis of the replication cycle of Orpheovirus

Abstract

BackgroundAfter the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated.MethodsIn this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays.ResultsWe observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant.ConclusionsThe results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Megaviruses contain various genes encoding for eukaryotic vesicle trafficking factors

“…Although the morphology and pathways of viral production processes vary somewhat among different giant virus lineages, factors encoded by the virus probably exploit the membrane trafficking routes of their host cells to ensure viral proliferation. That membrane trafficking plays a role during viral replication was also demonstrated by the addition of Brefeldin A, a drug that inhibits vesicle transport from the ER to the Golgi complex [29,30,44]…”

A Brief History of Giant Viruses’ Studies in Brazilian Biomes

“…), when cells stretch into a branched fusiform shape, and a late stage (starting at 24 h.p.i. ), when cells become rounded [64].…”

“…It is suggested that one or more particles of Orpheovirus, which are around 1.1 µm, are phagocytized by the host cell within 1 h.p.i. [62,64]. After entry, the particle's internal content is released when the membrane that surrounds the viral core fuses with the endosomal membrane through a structure called ostiole, located at the apex of the particle.…”

RNA Sequencing of Medusavirus Suggests Remodeling of the Host Nuclear Environment at an Early Infection Stage

“…Of note, the host nuclear membrane remains intact until near the end of the viral replication cycle, which represents a unique feature of medusavirus among currently characterized amoeba-infecting giant viruses. The viral replication cycles of other amoeba-infecting giant viruses are characterized as either a cytoplasmic replication by establishing cytoplasmic viral factories (e.g., mimiviruses [ 21 ], marseilleviruses [ 22 ], pithoviruses [ 5 ], cedratvirus [ 23 ], and orpheovirus [ 24 ]) or a nucleo-cytoplasmic replication, like in medusavirus, but with a degradation of the host nucleus (e.g., pandoraviruses [ 15 ] and molliviruses [ 1 ). For medusavirus, no visible cytoplasmic virus factory has been observed by transmission electron microscopy ( 2 ).…”