Podcast

Vinay Pathak, PhD, on Live-Cell Imaging of HIV-1

04/14/2021

In this podcast, Vinay Pathak, PhD, discusses the implications of his research presented in the session "Navigating to the Nucleus" at the Conference on Retroviruses and Opportunistic Infections 2021. The title of his presentation was "Live-Cell Imaging of HIV-1 Nuclear Import, Uncoating, and Proviruses."

Additional resource: 

Vinay Pathak, PhD, is a senior investigator and chief of the Viral Mutation Section of the HIV Dynamics and Replication Program at the National Cancer Institute in Frederick, Maryland. 

 

TRANSCRIPTION:

Leigh Precopio:  Hello everyone, and welcome to another installment of Podcasts360, your go‑to resource for medical news and clinical updates. I'm your moderator, Leigh Precopio, with Consultant360 Specialty Network.

Live‑cell imaging has drastically changed our understanding of HIV‑1 post‑entry replication events. Recently released research presented at the Conference on Retroviruses and Opportunistic Infections 2021 utilized live‑cell imaging to answer questions raised about nuclear import, retroviral uncoating, and proviruses in HIV‑1.

Today, I'm joined by Dr. Vinay Pathak, who is a senior investigator and chief of the Viral Mutation Section of the HIV Dynamics and Replication Program at the National Cancer Institute, and the presenter of this research at CROI 2021.

Thank you for speaking with me today. To begin, could you give us a brief overview of your session?

Vinay Pathak:  Certainly. Our recession was named "Navigating to the Nucleus," and it was organized and moderated by Dr Wesley Sundquist from the University of Utah, and Dr John Coffin from Tufts University.

This was a two‑hour on‑demand session that had 4 oral presentations that were focused on HIV replication in infected cells, and a potent small‑molecule inhibitor that binds to capsid and inhibits some of these replication steps. The talks were given by Dr Barbara Müller, myself, Dr Barbie Ganser‑Pornillos, and Dr Tomas Cihlar.

To give an overview of the subject, after fusion of the virus and infected cell membranes a viral capsid containing a genomic RNA enters the cytoplasm of the infected cell. After entering a cell, the virus has to accomplish two tasks. First, it has to reverse transcribe its RNA into a double‑stranded DNA and then bring that DNA into the nucleus so that it can integrate into the target cell genome.

We know that the capsid has to disassemble, a process that's often called uncoating, before integration can occur into the target cell genome. However, where and when these essential replication steps occur has been difficult to study, largely because the infected cells contain very few capsids. This makes it difficult to study using traditional biochemical methods.

In addition, it has been difficult to separate the behavior of infectious capsids from noninfectious capsids. The vast majority of the capsids that enter a cell are noninfectious, and don't complete the replication process. We estimate that only 1 in 50 virus particles that enter a cell lead to productive infection. So it's difficult to study the behavior of the infectious capsids from the vast majority of the noninfectious capsids.

Dr Barbara Müller gave an overview of the structure of the HIV-1 capsid, and the variety of methods used to image viral capsids in infected cells. She also gave an overview of their development of a new method of fluorescently labeling viral capsids for live‑cell imaging of assays.

My talk summarized the results of her studies using new labeling and imaging assays that were developed in my lab to study HIV replication in the infected cells.

Dr Barbie Ganser‑Pornillos gave a talk on the development of an efficient in vitro reverse transcription assay that was developed by her group and her collaborators, and their observations that intact are required for efficient reverse transcription.

Dr Tomas Cihlar from Gilead gave an exciting talk on the development of Lenacapavir, a small molecule inhibitor that binds to HIV-1 capsid, and potently inhibits viral replication. This compound inhibits HIV-1 replication at the molecular levels, is one of the most potent inhibitors of HIV-1 known so far and is currently being developed for therapy, as well as pre‑exposure prophylaxis.

Leigh Precopio:  Let's dive a bit deeper into your talk. How does live‑cell imaging influence the treatment and management landscape for HIV‑1?

Vinay Pathak:  As I mentioned above, there are very few HIV-1 virion infected cells and most of them are defective and do not complete replication thus making it difficult to study these events in biochemical assays. Recent studies have shown that live‑cell imaging of virions can overcome these limitations, and have provided some new and surprising insights into how HIV replicates in infected cells.

Live‑cell imaging can provide important new insights into the mechanism of HIV replication, and these insights can provide opportunities for development of a new classes of antiviral therapeutics.

Additionally, live‑cell imaging can provide insights into the mechanism of action of Lenacapavir and other potential antiviral drugs that target the viral capsid or other essential steps in the infected cells.

Finally, high throughput live‑cell imaging assays could help to identify new classes of small molecule inhibitors that interfere with the essential steps of HIV-1 replication, such as uncoating or nuclear import.

Leigh Precopio:  Your results indicated that infectious viral cores in the nucleus are intact and complete reverse transcription in the nucleus before uncoating, whereas it was previously thought that retroviral uncoating occurred in the cytoplasm. How will this deeper understanding of HIV‑1 nuclear import, uncoating, and other aspects of HIV‑1 post‑entry replication events that you studied impact future research in this area?

Vinay Pathak:  Our observations have raised many new questions about all aspects of how HIV carries out the essential steps required for replication in the infected cells, including the reverse transcription, nuclear import, uncoating, and integration.

The short‑term impact is likely to be that many groups will seek to verify and extend these operations to provide a more detailed understanding of the virus-host interactions that occur in the infected cells at the molecular level.

In the longer term, I think that an understanding of the virus-host interactions that are essential for replication, will provide new opportunities and targets for development of new classes of antiretroviral drugs.

Leigh Precopio:  What are the key take‑home messages from your session?

Vinay Pathak:  This was an exciting session and there were many take‑home messages. I will just mention a few. First, the technology for live‑cell imaging of virions is improving and evolving rapidly. We can expect that more efficient methods for specific labeling of virions will make these experiments easier and provide new insights into HIV replication.

A second take‑home message is that there was a paradigm shift in our understanding of how HIV-1 carries out essential steps in HIV replication. The change in our understanding has raised many new questions that I hope will be the subject of vigorous research in the coming years.

Third, it was exciting to hear that efficient in vitro reverse transcription was achieved after many decades of failed attempts by many scientists. Additionally, these studies prove convincingly that an intact capsid is required for efficient reverse transcription.

The fourth take‑home message is about the development of Lenacapavir as a therapeutic agent. This has been an amazing achievement. The high potency of the compound's long half‑life make it a very promising new anti‑HIV drug that could dramatically change the treatment and management of people living with HIV.

Leigh Precopio:  What are the next steps in this area of research?

Vinay Pathak:  This research has raised several obvious but important questions, each of which represent a large field of research.

First, how is an intact capsid imported through nuclear pores, into the nuclei of infected cells? Second, what are the molecular triggers that control the timing and efficiency of uncoating, and do host factors influence the uncoating process? Third, one of the host‑virus interactions that occur during the relatively short time period between uncoating and integration, that promotes efficient integration and viral gene expression? These and other questions should stimulate vigorous research and exciting new discoveries in the next few years.

So I think this was a very exciting session, and it shows how science can drive our understanding of even subjects that we knew very well such as HIV replication, and provide surprises in our understanding. That, as a scientist, is very exciting. I'm looking forward to future developments in this area of research.

Leigh Precopio:  Thank you for joining me today and answering all my questions.

Vinay Pathak:  Thank you so much. I appreciate the opportunity to talk to you today.