The progress of science depends on a continuing flow of detailed and reliable communications about the latest discoveries, observations and techniques. Big news in basic science is either held for a big conference or reported in one of the important science journals such as Science Magazine or Nature. But smaller reports of month-to-month progress are typically published in specialized journals that serve more specific fields of knowledge. For example, most virologists eagerly await each new biweekly issue of the Journal of Virology to see what their colleagues are up to.
This article takes a look at the September 2002 issue of the Journal of Virology to try to get a snapshot of the kind of HIV-related work deemed significant enough to be included. Of the 58 reports published in this issue, 13 specifically involve HIV. Other articles may concern SIV (which infects monkeys) or Feline Immunodeficiency Virus (which infects cats) as well as a number of other well-known viruses such as hepatitis C and B. Several of the AIDS-associated opportunistic viral infections such as CMV and HSV are represented, as are a host of esoteric but no doubt interesting viruses such as cauliflower mosaic virus and bovine viral diarrhea virus. But we're going to stick to HIV.
Keep in mind that most of the work published in this journal is more provocative than definitive. Usually the experiments were conducted in laboratory cell lines. Such in vitro studies may initially seem promising then later turn out to have little relation to what goes on in living beings. For the most part these papers are part of an ongoing discussion among workers in the field about what they have learned. Most of the reports were submitted in the Spring of 2002 and accepted at the beginning of Summer. It may seem insufferably geeky to want to examine HIV science at this level, but the need to know what's up is compelling.
One well-known innate antiviral defense system is the interferon response. If a cell becomes infected by certain kinds of virus, it will begin to secrete chemical messengers called interferons, which then travel through the bloodstream activating interferon receptors on uninfected cells. These activated but uninfected cells go through a series of changes that put them into an antiviral state. If the virus spreads to an interferon-primed cell, its protein-producing machinery stops dead in its tracks and the cell begins to die. This response means a dead end to any virus that tries to hijack that cell for replication.
One circumstance that can kick off the process of interferon secretion is the presence of double stranded lengths of RNA in a cell. Remember that DNA is the master molecule used to store an organism's entire genetic code within each cell's nucleus. A cell's genes are typically stored as coils of very long, durable, double strands of DNA composed of chains of nucleotides. Proteins, which are the stuff and substance of our bodies, are made from chains of amino acids. When a particular protein is needed, the gene for that protein is exposed and a temporary copy of the gene's DNA is copied onto a single strand of RNA. This is called transcription. This copy of the gene, called copy RNA (cRNA), is delivered from the nucleus to the cell's protein-making machinery where it becomes translated from a chain of nucleotides into a chain of amino acids. When translation is complete, the chain of amino acids folds itself up into a protein and gets to work. All together, this process is called gene expression.
HIV is somewhat unusual in that it keeps the genes for its proteins stored in a double strand of RNA instead of DNA. After HIV enters a cell, its RNA is copied into a strand of DNA by the viral enzyme reverse transcriptase. This piece of DNA is then delivered to the cell's nucleus and stitched into the master strand of DNA. Later, when the cell is stimulated to replicate, the viral genes are expressed right along with normal host genes and new virus particles are manufactured and released.
Although in some animals the presence of a double strand of RNA in a cell can trigger the interferon response, in plants and invertebrates, a double strand of RNA had been known to stimulate a different kind of response, called RNA interference. It seems that short pieces of double strand RNA have the remarkable ability to selectively and very efficiently thwart the expression of host cell genes that contain identical, complementary nucleotide sequences. It appears that the double strand RNA recognizes its cRNA twin, which leads to a shutdown of the protein-making machinery. In a classic experiment, a cell modified to produce firefly luciferase (a so-called reporter gene product that can be made to light up) was injected with pieces of double strand RNA transcribed from luciferase DNA. The result was literally like turning off a light.
Because the interferon response set off by double strand RNA is so powerful, RNA interference had never been observed in mammalian cells. A breakthrough came a couple of years ago with the discovery that double stranded lengths RNA shorter than 30 nucleotides did not set off the interferon response. Soon, through trial and error, it was found that a double strand of RNA in the neighborhood of 22 nucleotides long could effectively trigger RNA interference in human cells. These shorter bits of double strand RNA are called small interfering RNAs (siRNA). RNA interference is rapidly becoming one of the most important new techniques in the cell biologist's toolkit because it lets them switch genes on and off to see what they do. Being able to selectively "knock out" genes like this promises to revolutionize our understanding of how gene products interact in the complex environment of living cells.
The authors created small interfering RNA strands that corresponded to cRNA for segments of the HIV proteins Rev and Tat. They then inserted these siRNAs using transfection techniques into cells capable of supporting HIV replication. In every experiment, the cells dosed with siRNA showed dramatic and specific inhibition of HIV gene expression and replication. (In July, two groups also published reports of inhibiting HIV replication by inserting siRNAs targeting various HIV proteins.) Of course this raises a question of whether RNA inhibition can be used as therapy in people. The challenge will be to develop a way to safely introduce siRNAs into living cells. It may be possible, but such research is still in its very earliest stages.
There is also the possibility that RNA interference has been operating as a component of innate immunity all along. If so, then wily viruses like HIV may have already evolved a defense to this line of attack. While a therapeutic application of RNA interference may be viable down the road, in the meantime, virologists have a powerful new tool to use to tease apart the intricate web of protein-protein relationships that exists between HIV and its human host cells. What they discover may well yield the long-sought secret to defeating the virus.
Using transfection techniques to insert genetic material directly into cells in vitro, the researchers experimentally introduced DNA for HIV-1 along with either DNA for Caveolin-1 or an empty control into kidney cells. [Cells supplied with DNA inside the cytoplasm can process the genetic material and translate it into proteins. Genetic material can be directly injected or, more efficiently, packaged into an empty virus that is highly adapted to the job of introducing DNA or RNA into cells.] After the experimental transfection, the researchers then measured the amount of new HIV produced by using a reverse transcriptase activity assay. Unexpectedly, they found that while control cells continued to process HIV, nearly all HIV activity had been blocked in the cells expressing Caveolin-1.
They tried the experiment several other times to confirm their observation, and then tried the same experiment with several different laboratory strains of HIV. It still worked. Next, they tried the same experiment using a range of doses of Caveolin-1 and found that small amounts had a smaller effect than larger amounts, a convincing demonstration of activity called dose response. They also tried using a different cell type and using different ways of transfecting the cells, but the outcome was unchanged. To rule out the possibility that the inhibitory effect was due to RNA or DNA instead of the activity of the protein, they performed the experiment using a mutant piece of Caveolin-1 DNA that could not express the protein. This corrupted gene for Caveolin-1 failed to inhibit HIV, which supports the role of a functional protein. At this point they were convinced that transfecting Caveolin-1 along with DNA for HIV blocked expression of new virus, but how did it work?
Since a small amount of virus was still being expressed in the presence of Caveolin-1, they analyzed the HIV being produced to see if it could still infect other cells. It could, which suggested that although the amount of HIV expressed was dramatically lowered, the virus itself was not defective. They also did experiments to determine if the drop in viral production was due to a toxic effect of Caveolin-1 on the host cell. But again, the inhibitory effect seemed specific to HIV since the cells were still able to function normally. The case for HIV specificity was made stronger by an experiment showing that cells transfected with Caveolin-1 plus DNA for the measles virus did not inhibit the production of measles virions.
Finally, the researchers split the Caveolin-1 DNA into several fragments and tested each one until they found a segment of the protein that retained full activity. The segment, only about 34 amino acids long, corresponded to a region of Caveolin-1 that is normally buried within the lipid layers of cell membranes. Oddly, this part of the protein is not known to have any function other than interacting with itself or with another similar protein called Caveolin-2 (which Llano reports also inhibits HIV expression). Whether this protein exerts its effect on HIV directly or through intermediaries is unknown.
The author notes that HIV can replicate normally in cells that naturally express Caveolin-1 and speculates that some mechanism may compensate to create an environment permissive for HIV replication. Perhaps when Caveolin-1 is overexpressed, as in these experiments, the compensatory protein is overwhelmed. This is the perfect job for using RNA interference as a tool to tease out the interacting protein functions. Hopefully someone, somewhere, is busily turning genes on and off, looking for the one that unleashes Caveolin-1's HIV inhibitory potential.
Dendritic cells (DC) are often described as sentries patrolling the frontiers of the body, where outside meets inside along vulnerable mucosal tissues. DCs are mobile, and they regularly travel from the mucosal frontier to immunity centers in lymph nodes where they display samples of the invaders they've met at the gates. While some DCs express the CD4 molecule on their surfaces, suggesting that they can be targets for direct infection with HIV, they also express another cell surface protein than can bind HIV called DC-SIGN. It is thought that HIV can interact with DC-SIGN and either be internalized in the cell or simply carried along piggyback as the DC migrates to a lymph node. But this is like bringing a fox into the henhouse since it's in the lymph tissue that HIV meets up with its ultimate target, the T-cell.
Dendritic cells bearing the DC-SIGN molecule are found throughout rectal mucosa and in parts of the vaginal epithelium, two prime targets for blocking HIV infection with a topical microbicide. In their Journal of Virology article, Robert Doms and researchers from the University of Pennsylvania contribute a few new nuggets to our understanding of DC-SIGN and offer a new set of tools for future research.
First, the investigators report on a density analysis of DC-SIGN molecules on the surface of dendritic cells taken from seven volunteers over several months time. While there was some variability between and among individuals over time, the count of DC-SIGN molecules consistently exceeded 100,000 copies per cell. In previous studies using laboratory cell lines, the authors had determined that about 60,000 DC-SIGN molecules per cell was necessary to support efficient virus transmission.
The second part of their paper reported on a method of blocking DC-SIGN by using monoclonal antibodies (Mabs) to effectively reduce the number of molecules available for HIV attachment. Although the authors found a set of Mabs able to block virus binding in laboratory cell lines, these proved less effective at blocking virus transmission in a model using dendritic cells that had been stimulated from precursor cells. The authors propose that while partial inhibition of HIV transmission can be demonstrated by blocking DC-SIGN, there are most likely other cell surface molecules involved in binding and transmitting HIV. This paper offers some incremental knowledge about DC-SIGN and a new set of tools that may help researchers understand how HIV first interacts with the body. But as the authors admit, "our understanding of the factors that control DC-SIGN expression in vitro, and its pattern of expression on specific types of DCs in vivo, is far from complete."
Back to the GMHC Treatment Issues September 2002 contents page.