HIV’s Tat protein protects against RNA interference

Results of a test tube study published in the May edition of Immunity have shown that human cells use RNA interference (RNAi) to attack HIV. HIV, however, has evolved a counter-measure to prevent this activity and allow its replication in the body.

RNAi, or 'RNA silencing', is a primitive anti-virus defence mechanism that has been observed in plants and insects. It works by triggering the cell to break down RNA molecules produced from an infecting virus’s genetic material. This prevents the virus particles from reproducing themselves and spreading through the host.

In this study, researchers discovered for the first time that human cells also carry out RNAi in response to the presence of RNA sequences found in HIV's genome. HIV avoids being damaged by this mechanism by blocking the human cell’s RNAi activity using its Tat protein.

“HIV-1 elicits an antiviral defensive RNAi in human cells,” conclude the investigators. “Unless such induced RNAi is quelled by the virus through its Tat factor, viral replication may not proceed with success."

“The dynamic interplay between RNAi and suppressors of RNA silencing remains physiologically conserved from plants and invertebrates to higher vertebral animals,” they write.

RNA interference

RNAi is initiated by the presence of 'double-stranded RNA’ (dsRNA) molecules produced by virus particles in the cells they infect. These molecules are recognised by a protein called ‘Dicer’, which cuts the dsRNA up into small pieces called ‘small interfering RNA’ (siRNA). These fragments are then incorporated into a large complex of proteins called an RNA-induced silencing complex (RISC), which goes on to identify and degrade specific viral gene products in the cell.

The research team, from the United States National Institutes of Health and National Cancer Institute and from L’Institut de Génétique Humaine in Montpellier hypothesised that the dsRNA molecules encoded by the HIV’s genetic material could trigger RNAi in human cells. They searched databases of the genetic sequences of HIV, finding that HIV’s genome contains specific sequences that can form triggers for RNAi.

Although the genome contains many dsRNA sequences, they found that only one of these sequences has the ability to form the ‘hairpin’ shaped siRNA molecules necessary to trigger antiviral activity.

Despite the low levels of siRNA in each HIV particle, the investigators discovered that artificial forms of the siRNA molecule were able to inhibit the expression of HIV genes in human cells. This suggests that RNAi can occur in human cells in an attempt to reduce HIV replication.

HIV's counter-measure

As HIV can replicate successfully in the human body, the investigators postulated that the virus possesses a mechanism to counteract this antiviral effect. "If HIV-1 can induce sequence-specific siRNA silencing, then how does the infecting virus escape this pernicious restriction to replicate effeiciently in human cells?" they write. "One possibility is that, although the virus elicits RNA silencing, it also encodes a countervailing suppressor of RNA silencing."

By chance, the investigators found that HIV particles were susceptible to RNAi activity when Tat was not present in the test tube. In contrast, HIV particles reproduced themselves normally when Tat was present. Although known to be necessary for the initiation of HIV replication in cells, this suggested that Tat has an additional function of overcoming the human cell's antiviral acticity.

This was confirmed by comparison of the replication rates of normal HIV to virus containing a mutation in the portion of Tat that is responsible for overcoming RNAi. HIV with defective Tat replicated at a lower rate than ‘wild type’ virus.

“Intriguingly and unexpectedly, we found that HIV-1 evades elicited RNAi through a suppressor of RNA silencing function encoded in its Tat protein,” they explain. “Tat suppresses an otherwise effective restriction through functional abrogation of the cell’s Dicer activity.”

Therapeutic implications

The investigators point out that siRNAs are rare, being present in only one copy per HIV particle. The presence of siRNA is disadvantageous for the virus, as it triggers the human cell to fight against the virus. This leads the investigators to argue that it may be impossible for the virus to exist without the siRNA molecule, which would otherwise have been lost during evolution.

“The rarity of perfect duplexes in the HIV-1 genome suggests that this virus might have evolved sequence changes in order to escape ancient RNAi restriction,” they write. “We emphasise that currently we have no understanding as to the reason for this retention”.

If this conclusion is true, it could point the way towards a potential vulnerability of HIV to future treatment strategies. By blocking the protective activity of Tat, it may eventually be possible for the antiviral activity of the human cells to be unmasked. This may enable the human cells’ RNAi to fight off the virus and reduce HIV replication in the body without the virus being able to develop resistance against it.

Alternatively, the inability of this region of the HIV’s genome to change its sequence may make it a suitable target for an artificial RNAi-based anti-HIV strategy without the risk of the development of resistance.

However, the researchers also found that Tat had anti-RNAi activity not only for HIV-specific genes, but also for a range of genes from other organisms. This may have negative consequences for the future use of RNAi as anti-HIV therapy.

“Our results raise a challenge to proposed RNAi therapeutic strategies as applied to HIV-1,” they argue. Since Tat can inhibit the antiviral activity of RNAi, the use of artificial RNAi to treat HIV infection may need to wait until methods to overcome HIV’s counter-measure have been developed.