In Vitro Studies Revealed the HIV1 Replication Cycle

The AIDS virus can infect human T cells in culture, replicating itself and in many cases causing the lysis of the cell host (Figure 19-9). Much has been learned about the life cycle of HIV-1 from in vitro studies. The various proteins encoded by the viral genome have been characterized and the functions of most of them are known (Figure 19-10).

The first step in HIV infection is viral attachment and entry into the target cell. HIV-1 infects T cells that carry the CD4 antigen on their surface; in addition, certain HIV strains will infect monocytes and other cells that have CD4 on their surface. The preference for CD4+ cells is due to a high-affinity interaction between a coat (envelope or env) protein of HIV-1 and cell-surface CD4. Although the virus binds to CD4 on the cell surface, this interaction alone is not sufficient for entry and productive infection. Expression of other cell-surface molecules, coreceptors present on T cells and monocytes, is required for HIV-1 infection. The infection of a T cell, depicted in Figure 19-11a, is assisted by the T-cell coreceptor CXCR4 (in initial reports, this molecule was called fusin). An analogous receptor called CCR5 functions for the monocyte or macrophage.

After the virus has entered the cell, the RNA genome of the virus is reverse transcribed and a cDNA copy (provirus) integrates into the host genome. The integrated provirus is


Once the HIV provirus has been activated, buds representing newly formed viral particles can be observed on the surface of an infected T cell. The extensive cell damage resulting from budding and release of virions leads to the death of infected cells. [Courtesy of R. C. Gallo, 1988, J. Acquired Immune Deficiency Syndromes 1:521.]


Once the HIV provirus has been activated, buds representing newly formed viral particles can be observed on the surface of an infected T cell. The extensive cell damage resulting from budding and release of virions leads to the death of infected cells. [Courtesy of R. C. Gallo, 1988, J. Acquired Immune Deficiency Syndromes 1:521.]

transcribed and the various viral RNA messages spliced and translated into proteins, which along with a complete new copy of the RNA genome are used to form new viral particles (Figure 19-11b). The gag proteins of the virus are cleaved by the viral protease into the forms that make up the nuclear capsid (see Figure 19-10) in a mature infectious viral particle. As will be described below, different stages in this viral replication process can be targeted by antiviral drugs.

The discovery that CXCR4 and CCR5 serve as corecep-tors for HIV-1 on T cells and macrophages, respectively, explained why some strains of HIV-1 preferentially infect T cells (T-tropic strains) while others prefer macrophages (M-tropic strains). A T-tropic strain uses CXCR4, while the M-tropic strains use CCR5. This use of different core-ceptors also helped to explain the different roles of cy-tokines and chemokines in virus replication. It was known from in vitro studies that certain chemokines had a negative effect on virus replication while certain pro-inflammatory cytokines had a positive effect. Both of the HIV coreceptors, CCR5 and CXCR4, function as receptors for chemokines (see Table 15-2). Because the receptors cannot bind simultaneously to HIV-1 and to their chemokine lig-and, there is competition for the receptor between the virus and the normal ligand (Figure 19-11c), and the chemokine can block viral entry into the host cell. Whereas the chemokines compete with HIV for usage of the core-ceptor and thus inhibit viral entry, the pro-inflammatory cytokines induce greater expression of the chemokine receptors on the cell surface, making the cells more susceptible to viral entry.

HIV-1 infection of T cells with certain strains of virus leads to the formation of giant cells or syncytia. These are formed by the fusion of a group of cells caused by the interaction of the viral envelope protein gp120 on the surface of infected cells with CD4 and the coreceptors on the surface of other cells, infected or not. After the initial binding, the action of other cell-adhesion molecules welds the cells together in a large multinuclear mass with a characteristic fused ballooning membrane which eventually bursts. Formation of syncytia may be blocked by antibodies to some of the epi-topes of the CD4 molecule, by soluble forms of the CD4 molecule (prepared by in vitro expression of a CD4 gene genetically engineered to lack the transmembrane portion), and by antibodies to cell-adhesion molecules. Individual isolates of HIV-1 differ in their ability to induce syncytia formation.

Isolates of HIV-1 from different sources were formerly classified as syncytia-inducing (SI) or non-syncytium-inducing (NSI). In most cases, these differences correlated with the ability of the virus to infect T cells or macrophages: T-tropic strains were SI, whereas M-tropic strains were NSI. More recent classifications of HIV-1 are based on which coreceptor the virus uses; there is good but not absolute correlation between the use of CXCR4, which is present on T cells, and syncytia-inducing ability. The NSI strains use y Molecular Visualization Viral Antigens See Intioduction and HIVgp 120.

Kbp gag pol


vif vpu


Gene gag pol tat nef vpu vif vpr

Protein product

53-kDa precursor i p17 p24 p9 p7

16o-kDa precursor

gp41 gp120 Precursor

p64 p51 p1o p32

p14 p19

p27 p16

p23 p15

Function of encoded proteins

Nucleocapsid proteins

Forms outer core-protein layer Forms inner core-protein layer Is component of nucleoid core Binds directly to genomic RNA

Envelope glycoproteins

Is transmembrane protein associated with gp120

and required for fusion

Protrudes from envelope and binds CD4


Has reverse transcriptase and RNase activity Has reverse transcriptase activity Is protease that cleaves gag precursor Is integrase

Regulatory proteins

Strongly activates transcription of proviral DNA

Allows export of unspliced and singly spliced mRNAs from nucleus

Auxiliary proteins

Down-regulates host-cell class I MHC and CD4

Is required for efficient viral assembly and budding. Promotes extracellular release of viral particles, degrades CD4 in ER

Promotes maturation and infectivity of viral particle

Promotes nuclear localization of preintegration complex, inhibits cell division

FIGURE 19-10

Genetic organization of HIV-1 (a) and functions of encoded proteins (b). The three major genes—gag, pol, and env— encode polyprotein precursors that are cleaved to yield the nucleocapsid core proteins, enzymes required for replication, and envelope core proteins. Of the remaining six genes, three (tat, rev, and nef) encode regulatory proteins that play a major role in controlling expres sion; two (vif and vpu) encode proteins required for virion maturation; and one (vpr) encodes a weak transcriptional activator. The 5' long terminal repeat (LTR) contains sequences to which various regulatory proteins bind. The organization of the HIV-2 and SIV genomes are very similar, except that the vpu gene is replaced by vpx in both of these.


(a) Infection of target cell

(a) Infection of target cell

Visualizing Viral Replication

ssRNA Reverse ^^ \ transcriptase

RNA-DNA hybrid


{T) HIV gp120 binds to CD4 on target cell.

(2 Fusogenic domain in gp41 and CXCR4, a G-protein-linked receptor in the target-cell membrane, mediate fusion.

Nucleocapsid containing viral genome and enzymes enters cells.

Viral genome and enzymes are released following removal of core proteins.

Viral reverse transcriptase catalyzes reverse transcription of ssRNA, forming RNA-DNA hybrids.

Original RNA template is partially degraded by ribonuclease H, followed by synthesis of second DNA strand to yield HIV dsDNA.

The viral dsDNA is then translocated to the nucleus and integrated into the host chromosomal DNA by the viral integrase enzyme.

(b) Activation of provirus

(b) Activation of provirus

Activation Provirus

Transcription factors stimulate transcription of proviral DNA into genomic ssRNA and, after processing, several mRNAs.

(2 Viral RNA is exported to cytoplasm.

da Host-cell ribosomes catalyze synthesis of viral precursor proteins.

(3b Viral protease cleaves precursors into viral proteins.

HIV ssRNA and proteins assemble beneath the host-cell membrane, into which gp41 and gp120 are inserted.

(5a The membrane buds out, forming the viral envelope.

(5b Released viral particles complete maturation; incorporated precursor proteins are cleaved by viral protease present in viral particles.

FIGURE 19-11

Overview of HIV infection of target cells and activation of provirus. (a) Following entry of HIV into cells and formation of dsDNA, integration of the viral DNA into the host-cell genome creates the provirus. (b) The provirus remains latent until events in the viral particles. (c) Although CD4 binds to the envelope glycoprotein of HIV-1, a second receptor is necessary for entry and infection. The T-cell-tropic strains of HIV-1 use the coreceptor CXCR4, while the macrophage-tropic strains use CCR5. Both are receptors for chemo-

infected cell trigger its activation, leading to formation and release of kines, and their normal ligands can block HIV infection of the cell.

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CCR5, which is present on monocytes. Studies of the viral envelope protein gp120 identified a region called the V3 loop, which plays a role in the choice of receptors used by the virus. A study by Mark Goldsmith and Bruce Chesebro and their colleagues indicates that a single amino acid difference in this region of gp120 may be sufficient to determine which receptor is used.

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