Replication without integration; may be a key in unlocking cure
Replication sans integration?
HIV always seems to find a way around smartest strategies scientists cook up against it, and keeps coming up with surprises, the more researchers study it. Now cure researchers may well find that their understanding of how HIV hides in latently infected cells—the so-called HIV reservoir—may need to be adjusted, if a recent study is to be believed.
A quick review of the basics before we get into the study itself: HIV is a retrovirus, which means that once it enters its host cell, it produces a DNA copy of its RNA genome, and then integrates this DNA into the genome of the infected cell. Both reverse transcription and integration are believed to be required for retroviruses to replicate.
But now David N. Levy at the New York University College of Dentistry and his colleagues have found evidence that HIV might be an exception to this paradigm. The virus, it seems, can replicate even when its DNA does not integrate into the host cell genome, at least in cultured resting CD4+ T cells (J. Virol. 2013, doi:10.1128/JVI.01939-13).
In their study, Levy and colleagues treated resting CD4+ T cells with low amounts of cytokines such as interleukin 4. This made the cells infectible, without causing them to start dividing. They then infected the cultured cells with HIV, waited for five days, and activated them the same way they become activated when they encounter antigen: by stimulating their T-cell receptor.
This activation is known to induce HIV replication, and in this case, Levy and colleagues found that the activated cells could produce new viruses even when they prevented the viral DNA from integrating, albeit less efficiently: Each cell made about two thirds fewer HIV particles than when integration was intact. What’s more, the cells could still make HIV from unintegrated DNA if the researchers waited up to 30 days before activating them.
This suggests that like integrated DNA, unintegrated DNA can also serve as a persistent HIV reservoir. In principle, this mechanism might also operate in vivo, where HIV integration is not inhibited. That’s because Levy and colleagues found that infecting resting CD4+ T cells with normal HIV that can integrate also resulted in a sizeable fraction of cells that made HIV from unintegrated DNA. “If you don’t interfere with the integration process, this is still going on,” Levy says.
The unintegrated DNA in latently infected cells comes in two forms: linear or circular. Because the linear forms are believed to be degraded, Levy says, the most likely source for the HIV replication observed in the experiments is circular DNA.
Still, he adds, the results are likely going to be controversial, because some researchers believe that even the circular forms of unintegrated HIV DNA are unstable. While circles have been shown to be stable in non-proliferating cells in culture, he says, “there is intense controversy about whether they can persist in vivo.”
It’s also far from clear if HIV can also replicate without integration in vivo. “We are working on demonstrating that this can occur in an animal model in vivo,” says Levy, who plans to check whether latently infected cells taken from nonhuman primates infected with SIV (the monkey equivalent of HIV) can make virus particles from unintegrated DNA once the cells are activated.
Should the findings hold up in vivo, they could suggest unintegrated DNA as a source for the HIV reservoir in latently infected CD4+ T cells in addition to integrated DNA, Levy says. This wouldn’t necessarily mean that the reservoir is bigger than was previously assumed, he adds. But one implication, he says, is that studies of latently infected cultured cells that were infected in vitro need to take into account that a significant proportion of the observed viruses could be coming from unintegrated DNA.
Levy is also currently investigating whether the finding has implications for the favored strategy to eradicate viral reservoirs: inducing HIV replication in latently infected cells with drugs such as HDAC inhibitors, so that the infected cells die as a result of renewed virus replication or can be targeted by drug treatment or immune responses.
To this end, he is comparing the effects of drugs that are currently used to induce HIV replication from the integrated reservoir with their effects on the unintegrated reservoir. “[If] a piece of DNA is part of a chromosome or not, the rules about how it is turned on are likely different,” he says.
A quick review of the basics before we get into the study itself: HIV is a retrovirus, which means that once it enters its host cell, it produces a DNA copy of its RNA genome, and then integrates this DNA into the genome of the infected cell. Both reverse transcription and integration are believed to be required for retroviruses to replicate.
But now David N. Levy at the New York University College of Dentistry and his colleagues have found evidence that HIV might be an exception to this paradigm. The virus, it seems, can replicate even when its DNA does not integrate into the host cell genome, at least in cultured resting CD4+ T cells (J. Virol. 2013, doi:10.1128/JVI.01939-13).
In their study, Levy and colleagues treated resting CD4+ T cells with low amounts of cytokines such as interleukin 4. This made the cells infectible, without causing them to start dividing. They then infected the cultured cells with HIV, waited for five days, and activated them the same way they become activated when they encounter antigen: by stimulating their T-cell receptor.
This activation is known to induce HIV replication, and in this case, Levy and colleagues found that the activated cells could produce new viruses even when they prevented the viral DNA from integrating, albeit less efficiently: Each cell made about two thirds fewer HIV particles than when integration was intact. What’s more, the cells could still make HIV from unintegrated DNA if the researchers waited up to 30 days before activating them.
This suggests that like integrated DNA, unintegrated DNA can also serve as a persistent HIV reservoir. In principle, this mechanism might also operate in vivo, where HIV integration is not inhibited. That’s because Levy and colleagues found that infecting resting CD4+ T cells with normal HIV that can integrate also resulted in a sizeable fraction of cells that made HIV from unintegrated DNA. “If you don’t interfere with the integration process, this is still going on,” Levy says.
The unintegrated DNA in latently infected cells comes in two forms: linear or circular. Because the linear forms are believed to be degraded, Levy says, the most likely source for the HIV replication observed in the experiments is circular DNA.
Still, he adds, the results are likely going to be controversial, because some researchers believe that even the circular forms of unintegrated HIV DNA are unstable. While circles have been shown to be stable in non-proliferating cells in culture, he says, “there is intense controversy about whether they can persist in vivo.”
It’s also far from clear if HIV can also replicate without integration in vivo. “We are working on demonstrating that this can occur in an animal model in vivo,” says Levy, who plans to check whether latently infected cells taken from nonhuman primates infected with SIV (the monkey equivalent of HIV) can make virus particles from unintegrated DNA once the cells are activated.
Should the findings hold up in vivo, they could suggest unintegrated DNA as a source for the HIV reservoir in latently infected CD4+ T cells in addition to integrated DNA, Levy says. This wouldn’t necessarily mean that the reservoir is bigger than was previously assumed, he adds. But one implication, he says, is that studies of latently infected cultured cells that were infected in vitro need to take into account that a significant proportion of the observed viruses could be coming from unintegrated DNA.
Levy is also currently investigating whether the finding has implications for the favored strategy to eradicate viral reservoirs: inducing HIV replication in latently infected cells with drugs such as HDAC inhibitors, so that the infected cells die as a result of renewed virus replication or can be targeted by drug treatment or immune responses.
To this end, he is comparing the effects of drugs that are currently used to induce HIV replication from the integrated reservoir with their effects on the unintegrated reservoir. “[If] a piece of DNA is part of a chromosome or not, the rules about how it is turned on are likely different,” he says.
Comments
Post a Comment