Looking into the Eclipse
Authors: Joseph E. Rabinowitz, Jude Samulski
Journal: Molecular Therapy (2002)

Since childhood we have been told to never look into an eclipse. “Old school” virology demarcated the initial steps of viral entry after binding as the “eclipse phase,” in which critical but unmeasurable steps took place before successful infection could be determined (DNA replication followed by virus production). For the gene therapy vector adeno-associated virus (AAV), the eclipse phase has been defined as binding or adsorption, penetration, migration to the nucleus, and uncoating. The details of the eclipse phase are changing due to new research, headed by Christoph Bauchle at the Ludwig-Maximilian University in Munich Germany, using viral particles labeled with a single fluorescent dye [1Seisenberger G. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus.Science. 2001; 294: 1929-1932Crossref PubMed Scopus (701) Google Scholar]. Like a telescope on the far side of the moon, we are able to see into the eclipse phase of an AAV infection with great clarity. Unlike previous attempts to determine the normal behavior of AAV interacting with a human cell, now the view is unencumbered by scores of dye molecules or thousands of other virus particles competing to do the same thing. While only a coherent spot of light moving in a sea of static, the images, when viewed against the outline of the cell, tell a compelling story of viral infection (see http://www.single-virus-tracing.com). A single dye molecule is all that is needed to image each virion for up to 10 seconds before photobleaching. This window of time is opened at different points in the first 15 minutes after infection, revealing our first clear view ever into the eclipse phase. This study, which used imaging expertise from the Bauchle laboratory and viral expertise from the Hallek laboratory, has revealed the behavior of individual AAV virions in and around human cells. Over 1000 trajectories of AAV in the aqueous solution near the cell, in contact with the cell and releasing, in contact and penetrating, as well as within the cell tell a story that sheds new light on how this virus carries out the early steps of infection in human cells. We see single virus particles consecutively binding as many as 18 times before wandering out of sight. With only 13% of particles penetrating the membrane, even permissive cells act as a barrier to infection. So far, AAV vector preparations are produced with varying particle to infectious unit ratios. When tracing a single particle, it is not possible to determine if the data generated represent a noninfectious or infectious event. For this reason the authors measured over 1000 trajectories. It will be of keen interest if future development allows the tracking of a single virus through all steps of infection. However, the immediate promise of this technique will be how it resolves issues regarding viral binding, entry, uncoating, and nuclear transport. In a cell line defective for all proteoglycan biosynthesis, which includes heparin sulfate proteoglycans, the primary AAV2 receptor, there is a decrease in free diffusion that is similar to the decrease seen in cells with the normal compliment of proteoglycans. A general electrostatic attraction between the virus and cell may account for this reduction in free diffusion even in the absence of primary receptors. Regardless of whether the attraction resulted in touching and repulsion or touching and penetration, the amount of dwell time at the membrane was similar (62 versus 64 milliseconds). The tracings of trajectories that penetrated the membrane were fit into three types of motion: one was consistent with free virions, and the other two were consistent with the virus being within an endosome. One of the more interesting findings addressed a key question of viral entry: does AAV enter the cell through endosomes? Of the 43 independent tracings of viral penetration through the membrane into the cytoplasm, all were consistent with one virus within one endosome. This finding is quite similar to that of canine parvovirus entry, where one or two virions were seen in small vesicles at 15 minutes postinfection [2Basak S. Turner H. Infectious entry pathway for canine parvovirus.Virology. 1992; 186: 376-386Crossref Scopus (62) Google Scholar]. About half of the trajectories showed normal linear movement, either free or within an endosome, while just under half displayed hindered movement (endosomal), and 8% showed directed motion through the cytoplasm (endosomal). The latter movement was eliminated by nocodazole treatment, suggesting microtubule involvement. Taken together with previous work on AAV entry, a clearer picture now emerges: endocytosis of one or two AAV into clathrin-coated vesicles, whose movement may first be blocked by organelles or vesicle fusion, followed by directed motion, and finally the release from endosomes, which requires acidification [3Bartlett J.S. Wilcher R. Samulski R.J. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors.J. Virol. 2000; 74: 2777-2785Crossref PubMed Scopus (405) Google Scholar]. However, the cytoplasmic motion was not associated with any temporal data. Is there a normal progression of cytoplasmic movement of virus during the 15 minutes from linear, to hindered, to association with microtubule, to free diffusion? These questions can all be answered using this imaging technique. The speed of viral movement to the nucleus was surprisingly fast. Unlike a previous study that showed perinuclear accumulation at 2 hours postinfection when greater than 100,000 labeled virions per cell were used [3Bartlett J.S. Wilcher R. Samulski R.J. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors.J. Virol. 2000; 74: 2777-2785Crossref PubMed Scopus (405) Google Scholar], at the end of the 15-minute window, half of the cells showed trajectories within the nucleus. Also a surprise were the 34 tracings of particles that showed directed motion through the nucleus, which, like the same motion seen in the cytoplasm, were inhibited by nocodazole treatment. The explanation provided by the authors that these represent tubular structures continuous with the cytoplasm seems accurate, and these tracings should be considered cytoplasmic, not nuclear. It will be of interest to determine if this is a typical phenomena seen with all viruses, or if it is unique to AAV or the specific cell lines used in these studies. Although this study has allowed the field to gain great clarity into the eclipse phase of AAV infection, a question that has not been addressed is when the virions disassemble. The authors propose that by using different dyes to label the capsid and the genome one will be able to answer this question. Due to the size of AAV (20–25 nm) and other data, it has been suggested that the virus enters the nucleus before uncoating. From this study we do not know if the intact virion has entered the nucleus, but we do know that there is a nuclear localization signal on a minority of the capsid subunits that could potentiate this step [4Hoque M. Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation.J. Virol. 1999; 73: 7912-7915PubMed Google Scholar]. All of these points raise the interesting question of whether intact virions or subunits of the disassembled virion are responsible for the tracings within the nucleus. Finally, can the general research community adapt the “off-the-shelf” components articulated so well in this study to shed real-time insight on these and other interesting viral entry questions? Like all new technology, its “user friendliness” will determine if this will become a breakthrough technology for all labs interested in viral entry, or a technology that requires the support of a large department or center. Regardless, studies using this technique in the upcoming years may shed more light on the “eclipse phase” of viral infections than ever before.