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The Poxviridae family includes some of the largest DNA viruses known. While variola (the causative agent of smallpox) remains the most deadly member of the family, several other members, including monkeypox, tanapox, cowpox, vaccinia, Yaba-like disease virus and molluscum contagiosum, are capable of causing disease in humans. Orthopoxviruses, which include variola, monkeypox and vaccinia, have a double stranded genome of about 200 kb and are predicted to encode for approximately 200 functional open reading frames making them some the most complex animal viruses known. This complexity is best demonstrated during viral morphogenesis that results in a virion that is predicted to incorporate approximately 100 viral polypeptides and several morphologically distinct forms.
Viral replication occurs entirely in the cytoplasm in discrete areas know as viral factories and results in the first infectious form termed intracellular mature virions (IMV). A subset of IMV receives an extra double membrane wrapping derived from the trans-Golgi or endosomal cisternae and are referred to as intracellular enveloped virions (IEV). After wrapping, IEV are transported via microtubules to the cell periphery where the outer membrane of the IEV fuses with the plasma membrane depositing one of the newly acquired membranes into the plasma membrane and releasing the enveloped virion from the cell. Many enveloped virions remain attached to the plasma membrane and are termed cell-associated-virus (CEV). Viral proteins deposited into the plasma membrane, by the fusion of IEV at the plasma membrane, direct the polymerization of actin on the cytosolic side forming what are called actin tails, which serve to propel CEV away from the cell and towards adjacent cells. CEV released from the plasma membrane are termed extracellular enveloped virus (EEV). IEV, CEV and EEV make up the enveloped form of vaccinia virus and IMV are considered unenveloped. While IMV represents the majority of progeny virions they are not released from the cell making the enveloped form responsible for cell-to-cell spread. Presently only seven viral proteins have been found to be specific to the enveloped form, and of these seven only six have been shown to be required for efficient envelope virus production. The major focus of my laboratory is the study of poxvirus morphogenesis, emphasizing the intracellular envelopment process. We employ molecular virological techniques along with state of the art live video microscopy and cell biology to study viral egress with the goal of understanding the molecular mechanism employed by poxviruses to produce intracellular enveloped virions. Furthermore, our research should provide insight into such cellular processes as protein trafficking, membrane and vesicle formation and intracellular trafficking.
Accompanying movie to
Fig. 4. HeLa cells were infected with the virus designated in the text. The following day, B5-GFP was imaged in live cells using time-lapse fluorescent microscopy. Images were collected at 1 frame/sec and are played back here at 10 frames/sec. The first two arrows in the sequence depict virions trafficking away from the site of wrapping. The diagonal set of connected arrows points to the virion movement depicted in Figure 4 of the text. The final set of bracketed arrows shows a virion that displays both anterior and retrograde movement. Also depicted are the site of wrapping (SW) and the nucleus (N).
Quicktime movies showing the entire time-lapse sequence used for Figure 5.
Time ratio is 1:10. Scale bare = 5µm.