For more than 20 years, HIV biologists have puzzled over why resting, in contrast to activated, CD4 T-cells so effectively resist HIV infection. Is this highly effective block due to an active inhibitory mechanism, or simply the lack of a key factor or nutrient in these cells? If the former, it might open the door to an entirely new strategy for blocking the growth of HIV in cells where the virus normally grows well.

A3G–A New Player in HIV Biology
Recent studies in Dr. Warner Greene's laboratory at the Gladstone Institute of Virology and Immunology have shed new light on this question. As reported in the journal Nature, an active defense mechanism is involved, and a key player in this defense is an antiviral host factor termed APOBEC3G (A3G).

A3G is incorporated into budding HIV virions and induces lethal mutations during reverse transcription of the virus in the next infected cell. Deamination of deoxycytidines in the minus strand of the virus (the first strand synthesized) causes dG to dA mutations in the plus strand (the second strand synthesized). The minus strand containing dUs may also be more rapidly destroyed by cellular enzymes.

The A3G story began in 2001 with the molecular cloning of this factor by Dr. Ann Sheehy, a postdoctoral fellow working with Dr. Michael Malim at the University of Pennsylvania. Its isolation galvanized the field. Many laboratories rapidly joined the effort to understand how the anti-HIV factor works. In rapid-fire order, four groups published papers in Science, Nature, and Cell identifying A3G as a DNA mutator enzyme (more precisely a deoxycytidine deaminase). In brief, these studies revealed that A3G is incorporated into budding virions and exerts its antiviral action in the next infected cell by mutating nascent viral DNAs formed during the course of reverse transcription.

New Tricks from an Old Protein
However, HIV does not take these antiviral effects of A3G lying down. Instead, it dedicates one of its nine genes to neutralize the threat posed by A3G. The Greene laboratory was the first to describe how Vif overcomes A3G's antiviral effects. They showed that this viral protein both targets A3G for rapid destruction in the proteasome (a cellular “garbage disposal” that normally eliminates unwanted or expired proteins) and partially inhibits the synthesis of new A3G proteins. In combination, these two actions effectively deplete the intracellular stores of A3G, making it unavailable for incorporation into new virions no enzyme, no virion incorporation, no DNA mutation, and no antiviral effect.

Vif circumvents the anti-HIV action of A3G by depleting the intracellular stores of A3G in infected cells, making this antiviral enzyme unavailable for incorporation into the virions budding from these cells. Vif partially impairs the synthesis of A3G and targets the already synthesized enzyme for rapid degradation by the 26S proteasome. The simultaneous binding of Vif to A3G and to an E3 ligase complex produces polyubiquitylation of A3G, thereby “marking” the protein for accelerated proteasome-mediated degradation.

Vif-A3G is an exciting new target for anti-HIV drug discovery. Millions of small molecules could be rapidly screened in an assay developed in the Greene laboratory. This assay involves the coexpression of Vif and A3G tagged with green fluorescence protein (GFP) in cells. Normally, Vif induces proteasome-mediated degradation of the A3G-GFP fusion protein, leading to little or no GFP epifluorescence in the cells (1). A small molecule (X) that prevents Vif from binding to A3G (2) or recruiting the key cellular E3 ligase (3) would result in the accumulation of A3G-GFP within cells, causing them to turn green. A robotic high-throughput version of the assay could survey small-molecule libraries for active molecules.

This new level of understanding is already propelling an exciting search for Vif antagonists. Gladstone scientists have developed an assay that appears promising for identifying small molecules that either block the assembly of Vif and A3G or the recruitment of key host factors (such as the E3 ligase complex) that target A3G for destruction in the proteasome. Most investigators in the field regard the Vif–A3G axis as the most promising new drug target to emerge since the discovery of the HIV coreceptors. The key is to realize this goal.

But this was only the tip of the A3G iceberg. Dr. Ya-Lin Chiu and Kim Stopak in Dr. Greene's laboratory began exploring how A3G is regulated in cells. They found that the enzyme exists in both high- and low-molecular-weight forms in cells. Curiously, only the LMW form possesses detectable enzymatic activity. Further, the HMW form can be converted into the LMW form by treatment with RNase, suggesting the existence of a large inactive A3G complex containing a key but uncharacterized RNA component.

Is the LMW form of A3G in unstimulated CD4 T-cells responsible for the strong postentry block encountered by HIV in these cells? Activation of the CD4 T-cells might remove this block by recruiting A3G into the inactive HMW ribonucleo-protein complex (see shift induced by PHA/IL-2 and anti-CD3/anti-CD28 antibodies).

Of note, the HMW complex predominated in all of the A3G-expressing T-cell lines that were evaluated. But the plot thickened with the discovery that resting human CD4 T-cells and monocytes express only the LMW form of A3G. The story became even more interesting when T-cell activation by mitogens or select cytokines was shown to recruit LMW A3G into the HMW complex.

Similarly, when monocytes were induced to differentiate into macrophages, A3G again shifted into the enzymatically inactive HMW form. The fact that resting CD4 T-cells and monocytes are quite resistant to HIV infection while activated CD4 T-cells and macrophages are highly permissive for productive HIV infection raised the distinct possibility that the LMW form of cellular A3G plays a key role in mediating the long-recognized but poorly understood block to HIV replication occurring in resting CD4 T-cells and monocytes.

To test this notion, Dr. Chiu in the Greene laboratory introduced small interfering RNAs specific for A3G into resting CD4 T-cells to “knock down” A3G expression. However, a new technology was required because these cells are notoriously difficult to transfect by traditional techniques. Amaxa nucleofection technology rode to the rescue. Remarkably, when A3G expression in resting CD4 T-cells was knocked down by nucleofection of specific small interfering RNAs, these cells became readily infectable with HIV. This finding indicates that the LMW A3G protein actively produces a strong block to HIV infection in resting CD4 T lymphocytes.

The LMW form of A3G found in unstimulated CD4 T-cells plays a key role in blocking HIV growth in these cells. Activation of these cells relieves the block by recruitment of A3G into inactive HMW complexes.

Next, studies were performed to determine how A3G produced these effects. Examination of the kinetics of reverse transcription in resting CD4 T-cells revealed that A3G markedly slowed this key step in the viral life cycle. Next, sequencing reactions were performed to determine if the slowly produced reverse transcripts contained the signature mutations indicative of A3G action. These studies revealed only rare A3G-mediated mutations, suggesting that the mechanism of inhibition could not be explained solely by deoxycytidine deamination. Rather, it seems likely that the ability of LMW A3G to bind RNA may play an important role in restricting the function of the reverse transcription complex. The finding that incoming virions contain little if any Vif and the fact that Vif is only produced much later in the HIV life-cycle highlight another key feature of this postentry block: this antiviral function of A3G is very active against wildtype versions of HIV.

New Insights and New Possibilities
These findings provide at least part of the answer to the 20-year riddle of why resting CD4 T-cells are so resistant to HIV. LMW A3G is the key. This antiviral enzyme actively inhibits growth of the virus principally by delaying reverse transcription in the absence of overt mutation of the reverse transcripts formed. These studies raise an important question: Can we harness this new information to produce a novel form of antiviral therapy? One possibility would be to identify small molecules that disassemble the HMW A3G complex, thereby creating in highly permissive cells the same antiviral shield that protects resting CD4 T-cells. While exciting, we must consider the fact that the A3G is inserted into an inactive HMW complex for a reason, possibly to protect the cell’s own DNA from mutation during cell division.

In summary, A3G forms an exciting new focus in HIV biology. Scientists at the Gladstone Institute of Virology and Immunology have been deeply involved in elucidating the mode of action of A3G, and they will continue to be on the cutting edge of this and other studies that could eventually lead to novel HIV treatments.