How HIV infects human cells

PNAS: Welcome to Science Sessions, the podcast of the Proceedings of the National Academy of Sciences, where we connect you with Academy members, researchers, and policymakers. Join us as we explore the stories behind the science. I'm Paul Gabrielsen, and I'm speaking with Vinay Pathak of the National Cancer Institute in Frederick, Maryland. In a PNAS article published last year, he and his colleagues explored how the human immunodeficiency virus, or HIV, enters and infects a cell. They focused on the timing and location of the uncoating of the virus, when it sheds the protein coating surrounding the viral genetic material before integrating itself into the host genome. For making a significant contribution to their field, the study was awarded a 2020 Cozzarelli Prize in Biomedical Sciences.

Vinay, how did you become interested in HIV?

Pathak: I started in graduate school at the University of California, Davis, right about the time that people became aware that acquired immunodeficiency syndrome, or AIDS, was becoming alarmingly prevalent among gay men in the United States. The epicenter of AIDS at the time was San Francisco, which is just about an hour drive from UC Davis, where I was a student. I was also working at the time on mouse mammary tumor virus, a retrovirus that causes breast cancer in mice. So, at the time, I was immersed in an environment where everyone in our lab was tremendously interested in what is causing AIDS, whether it is a virus, and if so, what type of virus it is, and so on. Later on, it was discovered that AIDS is caused by HIV, a human retrovirus. So, I became even more interested in HIV and AIDS and then decided that one day, I'd like to work on HIV and do what I can to help with this AIDS pandemic.

PNAS: Tell us about how viruses infect cells. How does the process work generally?

Pathak: So, viruses by definition exist outside of cells but replicate inside cells. So, before a virus can replicate, it has to enter a cell. Viruses typically accomplish this by binding to a cell surface receptor protein, and after the virus enters the infected cell, it uses the host cell machinery to produce viral proteins and copy its genomic nucleic acid, RNA or DNA, to make more viruses that then exit the cell and go on to infect other cells to repeat the cycle.

So, HIV and other retroviruses package an RNA genome, so ribonucleic acid is packaged into the viral capsids. It uses the enzyme reverse transcriptase that's packaged inside the capsid to copy that RNA into a double-stranded DNA form. And then that double-stranded DNA is transported into the nucleus, where it integrates into the chromosomal DNA of the target cell. And once it integrates it's called a provirus. So, a provirus is a term for the viral DNA that is integrated into the whole cell genome.

PNAS: What is uncoating and why is it a key step in this process?

Pathak: So, HIV and other viruses typically have a protein shell called a capsid that surrounds their genetic material. Sometime during the viral replication process that capsid shell must be disassembled. And this capsid disassembly process is called uncoating. Why this is a key step may vary for different viruses. In some viruses, it may be necessary to disassemble the capsid so that the viral nucleic acid can be efficiently replicated. In other cases, the exact opposite may be true, that it may be necessary to keep the capsid intact so that efficient replication of the genome can proceed.

PNAS: Why is it important to know where and when a virus uncoats?

Pathak: Understanding where and when the virus uncoats can provide valuable insights into how the virus carries out its key essential steps in replication. For example, if the capsid disassembles after its genome is replicated, it might mean that the capsid ensures that the viral enzymes needed to copy the genome remain in close proximity to the genome. On the other hand, it might suggest that there's a mechanism to allow substrate nucleic acid triphosphates to enter the capsids so that the genome replication can proceed. Or additionally the capsid may shield the viral genome from being detected by the host cell nucleic acid sensors, which can trigger induction of interferons and innate antiviral defense mechanisms. So, overall, understanding where and when uncoating occurs increases our basic knowledge and understanding of how the virus carries out essential steps in its replication, and this knowledge in turn can help us identify new targets for antiviral drug development.

PNAS: What did we know about HIV uncoating before your study?

Pathak: Before our study, it was thought that the viral capsid is too large to fit through the nuclear pores to enter the nucleus, and therefore, uncoating must occur in the cytoplasm. It was also thought that viral DNA replication or reverse transcription is completed in the cytoplasm and the viral DNA and a few viral proteins associated with that DNA form a preintegration complex. A few studies had also proposed that uncoating occurs while the viral cores are docked at the nuclear envelope, and then the viral preintegration complex is released from this core and enters the nucleus.

PNAS: How did you identify the location and timing of uncoating?

Pathak: We developed a method to label viral capsids with green fluorescent protein without significantly affecting their infectivity. And this was an important technical advance. We could then follow these fluorescent capsids in the cytoplasm, through nuclear import, and into the nucleus. We found that the fluorescent capsids retain most of their fluorescent signals even after they entered the nucleus, which meant that they had retained most of the capsid protein even after entering the nucleus, and therefore we could conclude that the nuclear capsids are intact or nearly intact. After the nuclear entry of the capsids, we observed the fluorescent capsid signals abruptly disappeared in the nuclei, indicating that the capsids had disassembled or uncoated in the nucleus on average about 10 and a half hours after infection. It was also important to make sure that we were observing the behavior of the infectious viral capsids, which are a small minority of capsids that enter an infected cell. To identify the infectious viral capsids, we developed a method to visualize transcriptionally active proviruses by fluorescently labeling the viral RNA being transcribed from the proviral DNA integrated into the cell chromosomes. We found that the nuclear fluorescent capsids uncoated very close to the site of integration. Subsequently a transcriptionally active provirus appeared very close to the site of uncoating. And this observation enabled us to conclude that we were observing the uncoating of an infectious viral core since it led to the formation of a transcriptionally active provirus.

PNAS: Why is your capsid labeling method an important advance?

Pathak: People have tried to label the capsid before with fluorescent proteins and other tags, but they always led to capsids that were not infectious. So, those tags led to inactivation of the virus capsid and its infectivity. So, therefore, we couldn't study how it behaves when it's able to complete its replication. So, labeling itself was not new, but labeling in a way that retained the infectivity of the virus was an important step.

PNAS: What are the other potential applications of this method in virology?

Pathak: This provides a general method for us to study many aspects of the virus replication and infected cells. And theoretically this approach can be applied to understand the details of other aspects of virus replication, for example, things that we haven't looked at, such as what happens between uncoating and integration, and other steps in replication. So, it's a general method that can be used to study HIV-1 as well as other retroviruses, like HIV-2 or simian retroviruses or other retroviruses that cause disease. So, we will be able to apply the same approach to the study of other retroviruses.

PNAS: Why is this finding significant in understanding how HIV behaves in a host cell?

Pathak: So, I think the fact that the viral cores are intact in the nucleus and uncoat just before integration has a significant impact on our understanding of almost all aspects of the virus replication in the infected cells. For example, one of the important outcomes is that the traditional view was that HIV-1 uncoats and reverse transcribes its genome in the cytoplasm and completes reverse transcription in the cytoplasm. Now we know that the reverse transcription actually occurs within an intact capsid and, although it may be started in the cytoplasm, that it is completed in the nucleus. So, this has a very profound impact on our understanding of how our replication occurs. The other aspect is that we now know that intact capsids are imported through nuclear pores. And so this is quite new, and we don't know very much about how the capsid remains intact and gets through the nuclear pore complexes.

So, understanding the details of how this essential step in the replication is carried out, could provide new potential targets for antiviral drug development. So, in addition, we know that maintaining an intact capsid is very important for a successful viral replication. So, small molecule inhibitors that either prevent the uncoating process or trigger premature uncoating, could form the basis of new classes of antiviral drugs. So, we also want to understand whether keeping the capsid intact until just before integration occurs, does that help the virus evade the host nucleic acid sensors and prevent the triggering of interferons and other host defense mechanisms, which helps the virus to go on and complete its replication. So, these findings raise a lot of interesting questions and new understandings of how replication takes place.

PNAS: How do these results advance efforts to treat HIV infection?

Pathak: So, I did allude to the idea that the capsid could be an important target for antiviral drug development. In fact, one such compound called lenacapavir is a very potent inhibitor of HIV replication. It binds the capsid; we don't know exactly how it inhibits replication, but it's very potent. And this compound is currently being developed by Gilead as a long-acting inhibitor for antiviral therapy and possibly pre-exposure prophylaxis. So, our results may provide valuable insights into the molecular mechanism by which this compound inhibits HIV replication.

PNAS: How did you feel when you heard your paper had won a Cozzarelli Prize?

Pathak: So, we think PNAS is a very important and high-impact journal, and we know that a very small proportion of papers that are submitted get accepted for publication. So, we were actually thrilled and felt privileged when our paper was accepted for publication in PNAS. So, after that, our paper being selected to receive this highly prestigious Cozzarelli Prize in Biomedical Sciences is truly a great honor that's beyond anything we ever imagined. So, we feel this is a great honor that's beyond what we expected. So, we're very appreciative. I also feel humbled and honored to work with a team of such talented scientists, especially Ryan Burdick, who's spearheaded these groundbreaking studies.

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