Beyond deepening understanding of poxvirus infection and immune responses, the findings also provide essential insights into other research areas as well. For example, poxviruses are now being employed as vectors for vaccines and gene therapy, as well as oncolytics viruses that infect tumor cells used to treat cancer. In ongoing research, the team is now working to understand the contribution of different DNA sensors to detecting poxvirus infection. Anna Williams is a content specialist in the medical school's Office of Communications.
She writes news stories about basic science and clinical research, as well as events, education and faculty news. She holds a bachelor's degree in journalism from New York University. What do we know about the assembly and release of new poxvirus virions?
In , the Journal of General Virology had a review article Smith et al. See Figure 1 in this review for an overview of what is known concerning the final stages of poxvirus assembly and release.
What is an update on the entry process? The transition from one state to the next is each accompanied by hallmark events. The first few steps broadly encompass virus entry and loss of the wrapping membrane, early mRNA translation, capsid uncoating, DNA release, and initiation of DNA synthesis. The onset of DNA replication coincides with a switch to transcribing intermediate mRNAs and the encoded transcription and other factors [ 2 ] then promote a switch to making late mRNAs and the proteins required for virion assembly.
Although many details remain to be elucidated, it is apparent that virus-directed proteolytic processing reactions play an important role in driving this process forward. Poxvirus DNA replication progresses within cytoplasmic structures originally called Guarnieri bodies and now commonly called factories [ 6 , 7 ].
The factories enlarge with continuing DNA synthesis, and gradually adopt a more irregular appearance as cavities form containing viral mRNA and host translation factors [ 9 ]. In the later stages of the infection cycle a complex of late gene products including D13, A14, and A17, plus a collective of viral membrane assembly proteins, act to dismantle the surrounding ER membranes and produce crescent-shaped structures as substrates for the assembly of immature virions IV [ 10 ].
IV are then processed into mature virion MV. MV are the most abundant infectious species although a small subset will acquire more membranes from the trans-Golgi network [ 11 ] and exit the cell by fusion with the cytoplasmic membrane. Curiously, any DNA transfected into infected cells is also replicated within factories by the same poxvirus proteins [ 16 , 17 ] where it is repeatedly copied and recombined [ 18 , 19 ]. The inextricable connection between replication and recombination is explained by the fact that E9 and I3 also catalyze a recombination-repair reaction dependent upon single-strand annealing [ 20 , 21 ].
This recombination machinery would play a critically important role in repairing double-stranded breaks [ 22 ] and broken replication structures reviewed in [ 23 ] and aiding virus evolution [ 24 , 25 ].
However, previous research has detected some seemingly contradictory features of these reactions. Moreover, there is a significant difference in the timing of the appearance of recombinant gene products depending upon how the crosses are organized. The DNA from two genetically-distinct co-infecting viruses has to mix to permit recombination and such interactions cannot happen until two or more developing factories collide and then often only late in infection [ 27 ].
Our studies show that poxvirus factories are surrounded by a matrix of cytoplasmic constituents and that these components, like mitochondria and ER, could create an impediment that partially inhibits the mixing of different viroplasma. This, plus the properties of the viroplasm itself, create a mixing barrier that is expected to delay the timing and limit the extent of poxvirus recombination even though the recombination and replication machinery is still active late in the infection cycle.
Poxvirus late gene products play a key role in virion morphogenesis reviewed in [ 29 — 31 ] and so to examine what else was happening around these time points, we tracked the association of three virus proteins D13, A5 and B5 with nascent virions using super-resolution structured illumination fluorescence microscopy.
A5 is incorporated into the virus core early in morphogenesis and is present in all forms of virus. D13 serves as a scaffolding protein and associates with both viral crescents and immature virions. The D13 scaffold is lost as immature virions mature into MV and thus serves as a marker of the early steps in assembly and IV. Finally, B5 is an envelope protein that serves as a marker for another intracellular virus, a form bearing two additional membranes called wrapped virus WV.
These steps in assembly are summarized in Fig 1A. Virus assembly begins with formation of viral crescents that eventually form IV. During maturation, the scaffold that gives shape to both crescents and IV is cleaved and the brick-shaped MV is formed.
A fraction of MV migrate towards the trans-Golgi network where they obtain additional membranes white and incorporate other unique proteins. B Representative images showing the timing of the appearance of each viral form.
C An inset of selected regions imaged at 10 hr post-infection. The ring structures formed by D13 red and B5 white are seen associated with the A5 cores green. MV tend to aggregate in clusters at later times in infection. D Distribution of virus forms at different times post-infection and total numbers of virus particles analyzed. The figure shows data consolidated from 3 independent experiments and reports a number-weighted average of the distribution of morphogenic forms using a total of 10 cells per time point.
We could also measure the proportion of each form relative to the total number of A5-bearing particles in fixed cells imaged at different times after infection Fig 1D. New MV appeared in significant quantities shortly thereafter, although these were mostly found outside of factories, where they later formed large A5-tagged aggregates Fig 1C. These WV were localized toward the cell periphery, near the plasma membrane.
These observations showed that virus assembly reactions are well in progress at the times when recombinant genes are first being detected. Using cells pulse-labelled for 15 min and then fixed and imaged, we saw that the greatest amounts of EdU were incorporated at early time points 3—4 hr post-infection and that this label was localized within virus factories and sometimes the cell nucleus Fig 2A. The rate of EdU incorporation peaked between 3—4 hr post-infection and declined thereafter with a half-time for the decay rate of somewhat less than 2 hr.
These kinetics parallel, but lag 1—2 hr behind the kinetics of E9L gene expression [ 32 ]. A Fluorescence micrographs showing the sites of EdU incorporation throughout the viral life cycle.
Nascent factories can be labelled brightly with EdU early in infection, the rate of incorporation declines with time as the factories mature and adopt a more diffuse appearance. Images represent a projection of all z-stacks. B Quantification of EdU incorporation. The figure shows data acquired from three replicate experiments, averaging all signals acquired from 13—19 cells per time point.
The error bars show standard deviations. These studies exhibited a lot of cell-to-cell experimental variation Fig 2B so we also investigated whether this late DNA synthesis could also be seen at sites and in cells where intergenomic recombination was also detected. To do this, we used live-cell microscopy and two different fluorescently-tagged molecules to track the development of virus factories and to detect the formation of recombinant genomes [ 27 ].
Recombination was detected using two different co-infecting VACV, each encoding a partially-duplicated fragment of a DNA-binding form of mCherry fluorescent protein mCherry-cro. The reconstruction of the mCherry gene by recombination permits expression of a full-length fluorescent protein from an early-late promoter [ 27 ].
The DNA binding tag serves to concentrate the recombinant protein in a region near the site s of recombination. During the earlier stages of the infection many factories were seen to collide and mix Fig 3A and S1 Video.
At 6 hr post-infection EdU was added to the specimen and the infection then allowed to proceed for another 35 min before being stopped, fixed, and processed to detect any newly incorporated EdU. In this particular specimen, the first signs of recombination between two co-infecting particles i. After processing the sample to label the DNA and the sites of EdU incorporation, the grid marks on the dishes were used to relocate the cells that had been imaged during the live-cell portion of the experiment Fig 3B.
The factories that had merged and produced recombinant mCherry had also incorporated EdU, showing that the replication machinery was still present and active in factories during and even after when recombinant formation was first observed. The cells were pulse-labelled for 35 min with EdU starting at 6 hr post-infection and fixed a few minutes after the last time point. These experiments were completed a total of three times and this figure highlights the results of a single experiment.
A Still images acquired during the live-cell portion of the experiment. Traces of the recombinant mCherry-cro reporter protein were first seen at hr post-infection, but only became obviously visible at 6 hr arrow. B The cell that was tracked during the live-cell portion of the experiment was reimaged after processing to detect any incorporated EdU. Both EdU and recombinant mCherry-cro molecules seem to be distributed throughout the factories. VACV factories develop in an environment densely populated with rough ER and this could provide an impediment to factory-factory interactions [ 8 , 27 ].
To better characterize the spacial relationships between the various cell and virus structures at times just preceding when recombinants start to be detected, we infected BSC40 cells with VACV strain WR, pulse-labelled the cells with EdU for 15 min, and processed the fixed cells at 6 hr post-infection to detect the DNA and EdU as well as the ER marker calreticulin.
In this example Fig 4 several factories are seen at lower magnifications, surrounded by an extensive network of calreticulin-tagged structures. The projection of the whole-cell image from 49 z-stacks tends to obscure the separation between the cytoplasm and the viroplasm in these projected images Fig 4 , upper panels.
However, the images assembled from seven nm z-stacks showed clearly that at least four factory elements are separated from each other by calreticulin-rich materials in this region of the cell Fig 4 , bottom. That these are still sites of replication is illustrated by the associated EdU label. Insofar as calreticulin serves as a proxy for ER membranes, this analysis showed that the ER, and other associated cytoplasmic components, creates boundaries that separate regions of viroplasm in each factory.
The top row shows the infected cell at lower magnification and combines all of the z-stacks in a projected image. The bottom row shows a projection of 7x nm z-stacks, located in the approximate middle of the cell.
In these more highly magnified images one sees calreticulin-labelled channels separating at least four EdU- and DAPI-labelled factories. Calreticulin is a well-established ER marker [ 33 ], but the staining seen using fluorescence microscopy Fig 4 provided no information regarding the integrity of the ER membranes that are presumed to surround the virus factories.
Nor can one see what else might occupy these channels. To investigate this question further, we used transmission electron microscopy to image VACV-infected cells Fig 5. We first studied cells fixed at earlier time points 3.
This timing also precedes the appearance of the first recombinants by several hours. Another example, captured at 4. In this electron micrograph one begins to see what looks like juxtaposed viroplasm in places, with membrane remnants lying in between what might have been the point of contact Fig 5B.
One also sees mitochondria lying along the borders and in-between some factories. We also see some evidence that remnants of these membranes persist following factory collisions. Membranes, some double layered arrows , and mitochondria are commonly seen along the edges of these factories. At 4 hr post-infection B some factories have migrated to a position adjacent to the nucleus Nu and membrane fragments are sometimes also seen surrounding and within the factory.
The static images seen in Fig 5 provide no insights into the earlier history of the different virus factories. This makes it difficult to interpret the significance of the membrane fragments and other cell contents that separate different regions of viroplasm. To gain some insights into what events preceded the formation of these structures we used a combination of light and electron microscopy to track the development of the factories and then image the observed points of contact.
The infection was stopped within the next 5 min, and the cell monolayer processed for transmission electron microscopy TEM. Sections were acquired aligned as closely as possible with the same X-Y plane captured in the optical images. The two images were subsequently aligned, taking advantage of the distinctive patterns formed by the cells and their contents Fig 6B. Interestingly, one sees a variety of cytoplasmic components trapped between the two recently collided factories Fig 6C , including some mitochondria.
Elsewhere one sees more traces of fragmented cell membranes and there was no obvious sign of mixing of the viroplasm in the short time after the collision event. A projected image is shown. The cells were fixed and processed for TEM at post-infection. B Image correlation. The last fluorescence image left is shown at the same magnification as an image obtained by TEM centre.
The light and electron micrographs could be well aligned using the different factories as fiduciary markers right although it is not possible to perfectly align the images due to slight differences in the optical and TEM image planes. C Magnified view of the region surrounding the point of collision between two factories. A variety of cell structures are seen still separating the two factories including membranous debris and many clusters of mitochondria. Also seen in these images are a few crescent structures and IV characteristic of this time point.
This experiment was repeated but imaged using scanning rather than transmission electron microscopy Fig 7 and S3 Video. Hence the reversed contrast. However, the z-resolution of a spinning disk confocal microscope extends beyond 50 nm, which means that the representative stills from the live cell portion of the experiment cannot exactly represent 50 nm sections and this tends to confound the realignment process. In this example the boundaries between the two factories are more difficult to determine, although a disconnected trail of fragmented ER-like double-membrane structures can be seen lying in between the two structures Fig 7B.
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