<p><strong>Professor Michaela Rumlová, head of a research group of the National Institute of Virology and Bacteriology at UCT Prague (and Vice-Rector for doctoral studies), holder of a licensed patent &nbsp;for antiretroviral testing assays, and recipient of the Czech Science Foundation’s President’s Award for HIV research, has also become the principal investigator of the bilateral INTER-EXCELLENCE II project “</strong><i><strong>Cellular factors regulating the export and packaging of unspliced ​​retroviral RNA</strong></i><strong>”, starting this year. In this interview, she explains how retroviruses such as HIV “hack” the cell in order to export their own genome from the nucleus into the cytoplasm, and then to the site where it is specifically packaged into nascent viral particles, a key step in viral replication. Her team is collaborating with the NIH (National Institutes of Health) and Rockefeller University to elucidate mechanisms that may help reveal new vulnerable points in the retrovirus replication cycle while simultaneously clarifying related cellular processes.</strong></p><h5><strong>Could you describe your project and its main objectives?</strong></h5><p>Retroviruses, such as HIV, are viruses that carry genetic information in the form of RNA; however, after entering a cell, they are able to reverse transcribe it into DNA and integrate it into the host genome. In order to replicate, they must transport their genetic information—in the form of unspliced RNA—from the nucleus into the cytosol. There, it serves both as a template for the production of viral proteins and as the genome of new viral particles. This is highly unusual, as the cell very carefully controls which RNA molecules are allowed to leave the nucleus. Normal protein-coding cellular RNAs, known as messenger RNAs (mRNAs), must undergo splicing—a processing step in which certain segments are removed—before being exported. When this process fails, mRNA that retains these segments is held back in the nucleus as part of quality control and subsequently degraded. Retroviruses have managed to outwit this cellular control machinery, allowing their unspliced RNA to pass into the cytoplasm.</p><p>Our project focuses on identifying the cellular proteins that assist retroviruses in this step and on the mechanism by which protein complexes bound to viral RNA are remodelled during RNA transport. The goal is to understand how the virus uses cellular mechanisms to its advantage and how this pathway is linked to the subsequent fate of the RNA, i.e. to its translation and packaging into new viral particles.</p><h5><strong>Why is this export of unspliced ​​RNA so important for retroviral replication?</strong></h5><p>Because this RNA has a dual role for the virus. On the one hand, it serves as a template for synthesizing viral proteins in the cytoplasm; on the other, it represents the genetic material itself, which must be packaged into new viral particles. The virus must package its RNA genome in its unspliced form, as only this RNA contains essential sequences without which further viral replication would be impossible.</p><p><strong>Simply put: </strong>the virus needs to get a complete copy of its genetic instructions out of the nucleus. Once the viral RNA is in the cytoplasm, it can be used as a template for synthesizing viral proteins; and a portion of these RNA molecules is then selected and packaged into a new particle as a genome. We are interested in how the virus forces the cell to allow viral RNA that is, from the cell’s perspective, “unfinished,” that is unspliced, to leave the nucleus. We are also investigating how the cell and the virus together - albeit unintentionally from the cell’s perspective - determine which RNA molecule will be translated into proteins and which will be packaged as the genome. This is one of the key, yet still not fully understood, steps of retroviral replication.</p><h5><strong>What do you think it was about your project that impressed the Ministry of Education, Youth and Sports’ jury the most?</strong></h5><p>I believe the decisive factor was a combination of several elements. First, the reviewers were primarily interested in the scientific problem itself. We are investigating a mechanism that is essential for retroviruses, but still not sufficiently understood, which may also have broader implications for the regulation of cellular processes. In addition, the quality of the team, our strong collaboration with the<strong> NIH </strong>and <strong>Rockefeller University</strong>, and the use of modern methods that will allow us to track viral RNA in great detail also played a role. It was also important that the project has clear objectives and a realistic plan.</p><h5><strong>How will this research contribute to the development of new treatments for retroviral infections?</strong></h5><p>It is important to say that this is primarily basic research. Rather than developing a new drug directly, we are trying to understand, at the molecular level, the step that the virus cannot do without. Only when we fully understand the mechanism of these cellular processes and identify the essential factors for the export of unspliced ​​RNA and how they function, will we be able to better determine which interactions or mechanisms represent potentially vulnerable points. Similar mechanisms associated with the export of unspliced ​​mRNA also appear in some diseases, such as cancer, so I &nbsp;see the benefit of our research mainly in that it will refine our understanding of these pathological processes in the cell, and retroviruses help us do so. Therefore, our work on retroviruses not only shows us how the virus works, but also how the cell itself works.</p><h5><strong>What responsibilities does your UCT Prague team have? How does the collaboration with your American partners work?</strong></h5><p>At UCT Prague, we are conducting the project’s primary experimental work. Our research involves cell cultures, viral models, and various biochemical techniques ranging from immunochemistry to microscopy that allow us to track viral RNA and identify its cellular partners. In collaboration with colleagues from <strong>NIH, Frederick</strong>, we are developing single-particle tracking methods to observe viral RNA movement in living cells across both space and time. Furthermore, we are working with colleagues from <strong>Rockefeller University to employ iCLIP </strong>methods for the precise mapping of protein-binding sites on RNA. Both collaborations are valuable to us, because they are not just about data sharing, but also about the transfer of expertise and detailed know-how.</p><h5><strong>Who else is collaborating on the project?</strong></h5><p>At UCT Prague, we collaborate closely with the Department of Biotechnology and the Department of Biochemistry and Microbiology. Externally, we work with colleagues from the Institute of<strong> Organic Chemistry and Biochemistry of the CAS (IOCB Prague)</strong> and the <strong>Central European Institute of Technology in Brno (CEITEC)</strong>. Our research naturally necessitates the integration of diverse fields of expertise; it requires combining virology, cell biology, and biochemistry with advanced imaging and modern approaches to study RNA-protein interactions. This is exactly what makes our project capable of addressing complex questions that a single laboratory cannot tackle on its own.</p><h5><strong>Are there plans to continue the project? Have you brought on new collaborators, and are you looking to welcome new colleagues to the team?</strong></h5><p>Yes, definitely. In basic research, it often happens that one answered question opens up several more. High-quality, properly interpreted data provide a foundation you can build on — and that is what moves scientists forward. It is already clear that the topic of retroviral RNA export and packaging has broader implications and may reveal more general principles of how RNA functions within the cell. We also hope the project will be attractive to students, as it connects classical molecular biology, biochemistry, and virology with cutting-edge biotechnological methods. Students are already involved in the project as part of their bachelor’s and master’s theses.</p><h5><strong>What was the personal turning point that motivated you to pursue a long-term investigation into retroviral particles?</strong></h5><p>I first encountered retroviruses in the early 1990s during my diploma thesis, when I was working at IOCB Prague on HIV-1 protease inhibitors. At that time, AIDS could not yet be effectively treated, and retrovirus research had a completely different dimension. From the very beginning, I was fascinated by how such a relatively small and simple virus can outsmart evolutionarily highly optimized cellular processes and turn them to its own advantage. And that has never stopped fascinating me. From a scientific point of view, retroviruses are remarkable; just consider one of their enzymes, reverse transcriptase, without which no diagnostic or research laboratory in the world could function today. Without it, it would be impossible to diagnose RNA viruses like SARS-CoV-2, influenza, or HIV using RT-PCR tests, or, for example to produce recombinant human proteins in bacterial cells.</p>

Tracking unspliced RNA: Professor Rumlová studies how retroviruses “hack” cells

<p><strong>High-performance liquid chromatography (HPLC) remains one of the most widely used analytical techniques for separating, identifying, and quantifying compounds in complex mixtures. Successful HPLC method development depends not only on selecting the correct column and optimizing the mobile phase composition, but also on choosing the appropriate detector. Even a perfectly optimized chromatographic separation provides little value if the detection system cannot properly capture and interpret the analytes of interest.</strong></p><p>In many ways, the detector acts as the <i><strong>“vision system</strong></i>” of the chromatograph. It converts separated compounds into measurable electronic signals, allowing analysts to identify components and determine their concentrations. The choice of detector therefore has a major impact on sensitivity, selectivity, robustness, and the overall quality of analytical data.</p><p>This article provides an overview of the most common HPLC detection techniques, discusses their strengths and limitations, and outlines the most important criteria to consider when selecting a detector for a specific analytical task.</p><h3><strong>HPLC Detection Technologies</strong></h3><p>HPLC detectors are typically divided into two major categories:</p><ol><li>Specific detectors (solute-property detectors)</li><li>Bulk property detectors</li></ol><p>Specific detectors respond to a particular chemical or physical property of the analyte. Ideally, their signal is largely unaffected by changes in mobile phase composition. Common examples include UV/Vis detectors, fluorescence detectors, and mass spectrometers.</p><p>Bulk property detectors, on the other hand, measure differences between the pure mobile phase and the mobile phase containing analytes. These detectors are considered more universal because they respond to properties shared by many compounds. Refractive index detectors are among the best-known examples, alongside conductivity and light-scattering detectors.</p><p>Both detector categories have important advantages depending on the application, sample type, and analytical goals.</p><h3><strong>Common Detector Types Used in HPLC</strong></h3><p>A wide variety of detectors are available for HPLC systems, ranging from highly universal devices to extremely selective and sensitive technologies.</p><p>Common detector options include:</p><ul><li>UV/Vis detectors</li><li>Diode array detectors (DAD/PDA)</li><li>Fluorescence detectors</li><li>Refractive index detectors (RID)</li><li>Conductivity detectors</li><li>Evaporative light scattering detectors (ELSD)</li><li>Charged aerosol detectors (CAD)</li><li>Mass spectrometry detectors (MS)</li></ul><p>Each technology offers a different balance of sensitivity, selectivity, robustness, operational complexity, and cost.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Detector_overview_with_characteristics_Graphic_by_KNAUER_84b0dd30b1.jpg" alt="KNAUER: Detector overview with characteristics. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Detector_overview_with_characteristics_Graphic_by_KNAUER_84b0dd30b1.jpg 245w," sizes="100vw" width="1293" height="596"></p><h3><strong>Selecting the Right HPLC Detector</strong></h3><p>There is no universally ideal detector for all HPLC applications. The best choice depends on several factors, including analyte chemistry, matrix complexity, required detection limits, and instrument compatibility.</p><p>Careful detector selection is essential for obtaining reliable and meaningful analytical results.</p><p>The following considerations can help guide the selection process.</p><h4><strong>1. Chemical and Physical Properties of the Analytes</strong></h4><p>The chemical nature of the compounds being analyzed is one of the most important selection criteria.</p><p><strong>Questions to consider include:</strong></p><ul><li>Do the analytes contain chromophores?</li><li>Are the compounds naturally fluorescent?</li><li>What is their polarity?</li><li>What is their molecular weight?</li><li>Are they volatile or non-volatile?</li></ul><p>Compounds containing UV-active groups are often well suited for UV/Vis detection, while fluorescent molecules can benefit from the high sensitivity of fluorescence detectors. Non-UV-active compounds may require universal detection approaches such as CAD, ELSD, or mass spectrometry.</p><h4><strong>2. Required Sensitivity</strong></h4><p>Detector sensitivity determines the minimum concentration that can be reliably detected and quantified.</p><p>Routine quality control analyses often rely on UV/Vis detectors because they provide sufficient sensitivity for many applications while remaining relatively simple and cost-effective. However, trace-level analysis or highly complex matrices may require more advanced technologies such as:</p><ul><li>Fluorescence detection</li><li>Charged aerosol detection</li><li>Mass spectrometry</li></ul><p>These techniques offer significantly lower detection limits and greater analytical specificity.</p><h4><strong>3. Detector Linearity</strong></h4><p>Linearity describes how well detector response correlates with analyte concentration across a defined range.</p><p>A detector with strong linearity ensures accurate quantification and simplifies calibration procedures. Poor linearity can introduce quantification errors, especially when analyzing samples across broad concentration ranges.</p><h4><strong>4. Dynamic Range</strong></h4><p>Dynamic range refers to the concentration interval over which the detector can reliably measure analytes.</p><p>Applications involving samples with highly variable concentrations require detectors capable of maintaining accurate response over a broad range. A wide dynamic range reduces the need for repeated dilutions or multiple analytical runs.</p><h4><strong>5. Selectivity</strong></h4><p>Selectivity defines the detector’s ability to distinguish target analytes from other matrix components.</p><p>This factor becomes particularly important for complex samples containing many interfering substances. Highly selective detectors reduce the risk of overlapping signals and false positives.</p><p>Mass spectrometry is often advantageous when dealing with unknown compounds or highly complicated sample matrices because it provides structural information in addition to quantitative data.</p><h4><strong>6. Budget Considerations</strong></h4><p>Detector cost is another practical factor that laboratories must evaluate.</p><p>UV/Vis and refractive index detectors are generally among the more economical choices, making them attractive for routine analyses and standard applications.</p><p>More sophisticated technologies such as fluorescence detection, CAD, or mass spectrometry typically require:</p><ul><li>Higher initial investment</li><li>Additional maintenance</li><li>More complex infrastructure</li><li>Greater operational expertise</li></ul><p>However, the overall value of the detector should always be considered in relation to analytical performance requirements and long-term application needs.</p><h4><strong>7. Instrument and Mobile Phase Compatibility</strong></h4><p>The selected detector must be compatible with the operational conditions of the HPLC system.</p><p>Important parameters include:</p><ul><li>Flow rate range</li><li>Pressure limitations</li><li>Mobile phase composition</li><li>Solvent compatibility</li></ul><p>Some detectors are highly sensitive to mobile phase composition or gradient changes, while others tolerate a broader range of operating conditions.</p><p>Ensuring proper compatibility helps prevent baseline instability, signal interference, and reduced detector performance.</p><h4><strong>8. Destructive vs. Non-Destructive Detection</strong></h4><p>Another important consideration is whether the analytical workflow involves only detection or also preparative purification.</p><p>Non-destructive detectors allow collected fractions to remain intact after detection, which is highly beneficial for preparative chromatography and purification workflows.</p><p>Destructive detectors, by contrast, consume or alter the sample during measurement and are therefore more suitable for analytical applications where sample recovery is not required.</p><h4><strong>9. Reliability and Ease of Operation</strong></h4><p>Practical usability is often just as important as analytical performance.</p><p>Some detectors are designed for straightforward plug-and-play operation, while others require:</p><ul><li>Frequent calibration</li><li>Specialized maintenance</li><li>Dedicated gas supplies</li><li>Vacuum systems</li><li>Advanced operator expertise</li></ul><p>Laboratories should evaluate the available technical support, user experience, and maintenance capabilities before implementing more sophisticated detection technologies.</p><p>Reliable operation and manageable maintenance requirements can significantly improve laboratory productivity and reduce downtime.</p><p>Choosing the correct HPLC detector starts with understanding both the sample and the analytical objective. Rather than selecting a detector simply because it is available, laboratories should ensure that the technology aligns with the chemistry of the analytes, required performance characteristics, and workflow demands.</p><p>In many cases, combining multiple detectors within a single HPLC system can provide even greater analytical insight by delivering complementary information from the same chromatographic separation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/KNAUER_Flow_scheme_for_detector_selection_Graphic_by_KNAUER_486004045f.jpg" alt="KNAUER: Flow scheme for detector selection. Graphic by KNAUER." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_KNAUER_Flow_scheme_for_detector_selection_Graphic_by_KNAUER_486004045f.jpg 245w," sizes="100vw" width="1316" height="808"></p><p>Interested in learning more? Future articles will explore individual HPLC detection techniques in greater detail, including their operating principles, application areas, and practical advantages for different analytical workflows.</p>

A comprehensive guide for selecting the appropriate HPLC detector

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Tracking unspliced RNA: Professor Rumlová studies how retroviruses “hack” cells