Fig. 8. (a) Transmission electron micrograph of a giant multi-nucleated cell formed in vitro by the fusion of an HIV-infected transformed human T4 lymphocyte with other lymphocytes from the same cell line (magnified 1200 times). The syncytium is rapidly producing virus particles. (b) The budding of viral particles in (a) (magnifled 25,000 times). The transformed, or tumor, cell line shown here and developed by Robert Gallo produces large numbers of HIV particles without undergoing cytolysis and has therefore been instrumental in AIDS research. (Photograph by Kunio Nagashima, NCI-Frederick Cancer Research Facility.)

stimulation of the immune system must be related to their unusually large number of regulatory genes. These "extra" genes, which were either evolved independently by the virus or were pirated from the host's immune cells, appear to work in concert with the host cell's machinery and extracellular signals to limit or enhance viral gene expression as needed for survival of the virus. Table 3 lists the known regulatory genes in HIV, for which the detailed functions are only partially known.

The state of controlled viral replication is lost in all species of AIDS viruses when they are placed in tissue culture. Viral replication takes place rapidly in peripheral blood lymphocytes when stimulated artifically to divide. An infected T4 cell transcribes proviral DNA into several thousand copies of viral RNA, which serve as genomes for new virus particles and templates for production of viral proteins. The redirection of cellular machinery for the massive production of viral components leads to a loss of the normal protein synthesis required to maintain cellular integrity. In addition, the RNA genomes and viral proteins assemble into infectious virus particles, which, in

some instances, massively bud from the cell surface, thereby destroying the cell. A single infected cell may produce 500 to 1000 of these. Massive viral replication may occur in vivo as evidenced by detectable levels of viral antigens or infectious cell-free virus circulating in the serum of about half of the AIDS patients at various times during the course of the disease.

Detailed knowledge of the cellular factors controlling the virus life cycle

in monocytes and macrophages comes from in vitro studies of visna-maedi virus. The visna-maedi life cycle is highly dependent on maturational factors in these cells. Less differentiated monocytes are more difficult to infect and the viral life cycle stops after proviral DNA is transcribed into RNA. As monocytes age, they are more easily infected, and viral replication proceeds all the way to the production of viral proteins. This regulatory program as

sures that viral proteins are produced at exactly the same time that monocytes mature into wandering macrophages and can thus interact with additional cells of the host immune system that are susceptible to infection.

Additional evidence for the interaction of viral regulatory genes and cellular factors comes from work in my laboratory. We have been developing sensitive, quantitative, in vitro assays of both the infectiousness of the AIDS virus and the effectiveness of antibodies or antiviral agents against the virus. Initially, we screened many human tumor T4 cell lines for their ability to become infected readily with HIV. We then used cell cloning strategies to select those that grow in uniform monolayers and then react to viral infection by rapidly fusing with nearby cells. Thus, when infectious virus particles are introduced onto the monolayer, each cell that becomes infected fuses with other cells to form a large, distinct single cell with many nuclei, called a syncytium (Fig. 7). The number of infectious viral events can be determined directly by simply counting the number of these large cells (Fig. 8). On refining this assay we noticed that initially no new virus particles were produced by infected cells during the cell fusion process-they were produced only after the cells had exhausted their ability to fuse. These observations suggested that the virus has a two-stage strategy for assuring its persistence in the immune cells of the host (Fig. 9). Once inside a cell the first stage causes the virus to direct its effort toward cell fusion. This mechanism accomplishes the business of spreading in the host without direct exposure to the antiviral actions of the host's immune system.

What are some of the details of this first stage? The viral regulatory genes within an infected lymphocyte or macrophage seem to be chemically connected to the host cell's normal surface receptors, for example, any of the

FORMATION OF SYNCYTIA IN MICROASSAY

Fig. 9. Photomicrographs of sequential stages of cell fusion and syncytial formation in the quantitative HIV I-induced infectivity microassay. The top picture depicts normal uninfected cells forming a monolayer. The middle and bottom pictures demonstrate cell-tocell fusion. Note the cell nests or clusters (arrow) that occur by day two in culture. By day four or five, these cell nests form the typical syncytia described in the text and shown in Fig. 8.

lymphokine, or cell-recognition, receptors, that is, the MHC receptors. These cell-surface receptors receive chemical signals that direct the cells around the body and induce normal immune activity in the lymph system. Normal signals that prepare the various neighboring immune cells to interact with each other seem to activate the HIV envelope genes within the infected cell to produce the "fusigenic" envelope proteins, gp120 and gp41, that cause cells to stick together and fuse.

What of the second stage? It seems that if the HIV-infected fusigenic cells fail to find neighboring cells susceptible to fusion, a new set of cell-membrane signals induces the viral genes to redirect the cell's machinery toward producing the additional structural components required to assemble new infectious virus particles. The massive production of these new particles, sometimes at the expense of the cell, can be considered a terminal last ditch effort on the part of the virus to infect new cells and thus survive in the host. As virus particles bud from the cell, they strip off pieces of the protective cell membrane. Normally, cell membrane components are constantly being re-formed through protein synthesis to keep up with the everyday import and export of cellular

TWO-STAGE MODEL OF VIRAL REPLICATION

Fig. 10. HIV regulatory genes, in response to extracellular signals, seem to produce two distinct stages of viral replication that assure survival of the virus in the host. Stage 1. In the presence of CD4-positive cells, the infected cell produces the fusigenic viral proteins, gp41 and gp120, that cause fusion of the infected cell's membrane with the membrane of a neighboring CD4-positive cell. The viral genome and reverse transcriptase is then transferred to the uninfected cell. The newly infected cell may then separate to become a latent infected cell. Alternatively, it may take part in the fusion process with other nearby CD4-positive cells to form a giant multi-nucleated cell called a syncytium. In this way, the infection spreads slowly with no interference by the immune surveillance system. Stage 2. When no uninfected CD4-positive cells are nearby, the syncytium switches into a state of uncontrolled viral replication, which produces thousands of new infectious virus particles. As these bud from the surface, they tear, or lyse, the membrane and thereby destroy the giant cell. A single latent uninfected cell, when stimulated by extracellular signals, may also undergo uncontrolled viral replication, resultIng in lysis of the single cell. The infectious viral particles now encounter immune defenses as they travel through the body to find new infectible cells.

materials. However, the uncontrolled production of 500 to 1000 particles per cell and the holes they create as they bud from localized areas on the cell surface, cause the cell to take on excess extracellular fluids, burst, and die.

The newly created HIV particles, unlike some other viruses, appear to undergo a relatively rapid predetermined decay caused by the spontaneous shedding of the gp120 molecule, the molecule that binds to CD4. The shedding is apparently due to the in

teractive yet weak protein structure of gp120. Studies in my laboratory show that the shedding takes from 8 to 15 hours. Hence a race begins to find a new infectible host cell before the virus particle loses its ability to infect. (See "The Kinetics of HIV Infectivity" for a detailed discussion of this process in vitro.) In summary, if the infected cell is locked in a compartment of the body with no direct access to infectible cells and therefore no chance for fusing, the virus programs the cell to produce hundreds of virus particles, which can rapidly diffuse in extracellular fluids or in the bloodstream.

Biological Properties of the Virus. Having discussed regulatory processes that help assure presistence of the virus, we now turn to structural properties that help the virus escape from host immune defenses. The glycoproteins gp120 and gp41 forming the envelope of HIV have two biological properties important to the survival of the virus: 1) they contain large amounts of carbohydrate (sugar), which serves to minimize and hide their protein binding sites from the host immune system and 2) they insert themselves next to or within the host cell's own self-recognition proteins, such as the MHC molecules. Both properties help the virus to escape from normal antiviral immune mechanisms previously outlined in Fig. 5.

Antibodies are Y-shaped proteins that neutralize the virus by binding to specific molecular protein shapes, called epitopes, on the viral envelope proteins (Fig. 10). In most lentiviruses, almost all neutralization epitopes are highly glycosylated (sugar coated), and these carbohydrate moieties completely conceal neutralization epitopes from immune recognition. As a result, the B lymphocytes are not able to produce highly effective neutralizing antibodies. In the case of HIV, we are a bit more fortunate in that some effective neutral

izing antibodies are produced (see “The Search for Protective Host Responses"). In all strains of lentiviruses, some epitopes are variably exposed and induce the production of neutralizing antibodies of very narrow specificity (that is, they recognize only the one viral strain). While the neutralizing antibodies may be effective against the original virus, mutations occur frequently in the genes for the viral envelope and lead to production of new virus particles with rearranged neutralization epitopes. The new particles now escape neutralization by antibodies. This process has been called antigenic drift, a term previously coined for influenza viruses, which cause the common cold. The mutational phenomenon is seen in sheep infected with visna-maedi virus, in horses infected with the EIA virus, and in humans infected with HIV.

Moreover, non- or poorly-neutralizing antibodies can facilitate the infection of macrophages. The loosely associated virus-antibody complex sticks to an antibody receptor present on the surface of the macrophage. The macrophage then engulfs the virus-antibody complex and thereby becomes infected (Fig. 10). Thus, certain antiviral antibodies produced during the lentiviral infection serve no useful biological purpose and therefore seem to perpetuate rather than eliminate infection in the host.

As previously mentioned, after a host cell has become infected, the viral glycoproteins insert themselves strategically next to or within the MHC antigens normally present in the cell membrane. Since MHC proteins are precisely the surface antigens that cells of the immune system use to recognize each other as self, the viral glycoproteins act very much like a "wolf in sheep's clothing." The net result is a form of molecular mimicry. In particular, recall that the presence of gp120 in the membrane of the infected cell allows it to fuse with any neighboring cell that has a