The Life Cycle And Molecular Biology Of Htlv1 And Htlv2

HTLV-1 and HTLV-2 are genomically and structurally typical of type C oncornaviruses (Figures 12-4 and 12-6). The integrated proviral DNA contains redundant long terminal repeat (LTR) sequences that contain enhancer and promoter elements for viral RNA transcription, the RNA transcriptional start and polyadenylation sites, and, at the 3' end of the transcribed RNA, a stem-loop structure termed the Rex Response Element (RRE). Like all retroviruses the HTLV contain genes, termed gag, pol, and env, that encode for critical structural and functional proteins. However, as the case with other complex retroviruses (e.g., the lentiviruses and foamy viruses) the PTLV/BLV genus also contains genes for proteins that regulate viral expression. Also, as is the case with other retroviruses, HTLV-1 and 2 have evolved to make maximal use of their approximately 9-kb genome by employing multiple RNA splicing patterns of their primary RNA transcript and differential and shifting start sites for protein translation.25

The life cycle of a typical retrovirus is shown in Figure 12-2. Like all retroviruses HTLV replication is thought to be initiated by the binding of the surface envelope protein gp46 to a specific cell surface receptor. Studies indicate that HTLV-1 and HTLV-2 share the same cell surface receptor, which differs from that of BLV and HIV.26,27 HTLV receptor(s) have not been definitively established, although two candidate molecules have been identified that may play this role. One is a 71-kd heat-shock-like

Figure 12-6. Organization of the HTLV proviral DNA and the major cognate polyproteins and cleaved structural, functional, and regulatory proteins. Each gene and protein is discussed in the text.

protein, while the other is a 32-kd protein encoded on the distal arm of chromosome 17q.28-30 Both viruses can infect many different hematopoietic, epithelial, and mesenchymal cell types in vitro, but analyses indicate that, in vivo, most HTLV-1 or HTLV-2 DNA is found in CD4+ or CD8+ T lymphocytes, respectively.31

HTLV virions or purified HTLV surface envelope proteins have been shown to activate human T lymphocytes. Our own data demonstrated that a monoclonal antibody directed against the 32-kd putative HTLV cell surface receptor also activated human T lymphocytes (Choi et al., in preparation). Because activation of target host cells facilitates retroviral reverse transcription and subsequent viral integration of double-stranded DNA into host cellular DNA, it would seem that HTLV has evolved to exploit a heretofore undescribed T-cell activation cascade, in part perhaps explaining its T-cell tropism.

Despite this advantage, however, comparative in vitro experiments indicate that cell-free HTLV virions replicate a thousandfold less efficiently than HIV virions.32 Our own experiments indicate that HTLV virions contain considerably less surface envelope protein per virion. Also, relative to HIV, HTLV reverse transcription is less robust, with the major difference observed in vitro being the "jump" from minus stranded "strong stop" DNA to plus strand first strand DNA synthesis (Choi et al., in preparation) (Figure 12-3). AH of the above may explain the lack of clinical evidence for in vivo HTLV cell-free viral transmission.16

Relative to HIV, HTLV reverse transcription is considerably less error prone due to the greater fidelity of its RNA-depend-ent DNA polymerase.33,34 There are no known "hot spots" for proviral DNA integration and there are no recognizable oncogenes associated with the HTLV-1 genome.7,9,16 Most infected cells contain only one integrated copy of viral DNA, which can be on any human chromosome with seemingly random or chaotic flanking host cellular DNA sequences. However, cultured human cells and some fresh tumor specimens have been shown to contain multiple HTLV integrants. Also, defective copies of HTLV have been identified in vivo and in vitro wherein either whole or portions of genes (e.g., pol) are missing from the proviral DNA or stop codon mutations result in aborted translation of viral proteins.22 It is speculated that the presence of defective copies of HTLV could convey a worse clinical prognosis in that, while such genomes would still retain the genes that encode the transforming properties of the virus (vide infra), they would not express all of the major immunogenic epitopes of the virus and thereby escape immune surveillance.

The gag gene of the HTLV encodes for three primary proteins (for simplicity we will use the terminology for HTLV-1 although there are subtle size differences between HTLV-1 and HTLV-2 proteins) (Figures 12-4 and 12-6). The p19 protein, which is myristoylated at its amino terminus, anchors the organizing virus replication complex at the interior of the cell surface and facilitates budding of virus particles. The p24 protein forms a capsid in the interior of the virion, which surrounds and protects the ribonucleoprotein complex, which is organized into a preferred three-dimensional structure in part by the gag p15 nucleobinding protein.

A translational frameshift allows for the pro-pol polyprotein to also be translated off of the primary RNA transcript (Figure 12-6). The aspartate protease protein functions to cleave the initial gagpolyprotein into its individual structural components leading to virus maturation and infectivity. The pol protein contains the RNA-dependent DNA polymerase and RNAse-H activity critical for reverse transcription and the integrase activity.

The envelope protein is translated as a polyprotein off of the singly spliced RNA message (Figure 12-6). The polyprotein is cleaved into the individual glycosylated surface protein gp46 and the transmembrane protein gp21. While the surface gp46 determines tropism by its binding to the cell surface receptor, the transmembrane gp21 facilitates entry into the target cell via either direct fusion and/or endocytosis.

The multiply spliced RNAs encode for two (and possibly more) key regulatory proteins, Tax and Rex, which modulate HTLV RNA expression and splicing, respectively7,35,36 (Figure 12-6). Upstream from its TATAA box promoter sequence, the HTLV LTR region contains three 21-bp imperfect repeats that indirectly interact with the Tax protein to trans-activate HTLV RNA transcription37-39 (Figure 12-7). These TAX responsive elements (TRE) have homology to the human cyclic AMP response element (CRE) sequence and TAX activation of viral RNA transcription has been shown to be mediated indirectly by its interaction with cellular CRE binding protein (CREB) (Figure 12-7). Early in HTLV infection most viral RNA is multiply spliced.40 As more TAX protein is produced, more multiply spliced RNA is produced, eventually leading to increased production of REX protein.

REX is a phosphoprotein localized specifically in the nucle-oli of HTLV-infected cells. REX is required for the production of two incompletely spliced viral mRNAs that encode either GAG, POL, or ENV products.41 REX function depends on the presence of a 5' splice donor (SD) signal in the beginning of ENV mRNA and in the 5' LTR for the GAG/POL mRNA as well as the RRE. The 5' SD seems to destabilize the mRNA in the absence of REX. REX interacts with the RRE to suppress splicing directly or by accelerated transport of the viral RNA into the cytoplasm, either by interference with the splicosome complex, opening specialized RNA transport, or possibly by stabilizing target RNA molecules (or both). REX may also have a second function in addition to regulation of spliced mRNA. At high concentrations of REX a down-regulation or inhibition of TAXmediated trans-activation occurs, providing further regulatory control of virus expression and possibly secondary effects on cellular RNAs.42

There are three growth phases in the life cycle of HTLV. In the early phase, the 5' LTR supports low-level transcription and, because the production of REX is low, the fully spliced regulatory mRNAs are primarily produced. In the mid-phase, increased levels of TAX stimulate overall viral RNA transcription, while REX retards the RNA splicing such that all the viral structural and functional proteins are produced. In the late phase the increased production of REX decreases the production of TAX and REX and decreases the trans-activation of

Figure 12-7. Tax mediated trans-activation of viral transcripts and host cellular genes. Tax in concert with CBP facilitates the binding of CREB to the TRE located in the HTLV LTR resulting in a marked increase of viral RNA transcription. Similarly, Tax can trans-activate a number of cellular genes (e.g., c-fos, c-erg-1, or IL-2R-a) via its interaction with a number of cellular transcription factors (e.g., CREB, serum responsive factor [SRE], or NF-kB). Tax also affects NF-kB regulated transcription in a less direct manner. IkB binds to NF-kB dimers in the cytoplasm and prevents their nuclear localization. Inhibitor of kB kinases (IKKs) phosphorylate IkB causing it to dissociate. NF-kB can then migrate to the nucleus and activate transcription of cellular genes while IkB is degraded via the proteosome pathway. HTLV Tax is known to stimulate the activation of NF-kB at several levels in this pathway. Tax acts to free NF-kB from cytoplasmic constraints by causing its dissociation via the activation of IKKs and by acting as a molecular chaperone for proteolytic degradation due to its ability to physically associate with both the phos-phorylated form of IkB and the proteosome. By this mechanism, Tax would be able to guide IkB to the proteosome to promote its degradation and prevent further association with other cytoplasmic NF-kB dimers. Tax also enhances the trans-activating capabilities of NF-kB by stimulating its dimerization.

Figure 12-7. Tax mediated trans-activation of viral transcripts and host cellular genes. Tax in concert with CBP facilitates the binding of CREB to the TRE located in the HTLV LTR resulting in a marked increase of viral RNA transcription. Similarly, Tax can trans-activate a number of cellular genes (e.g., c-fos, c-erg-1, or IL-2R-a) via its interaction with a number of cellular transcription factors (e.g., CREB, serum responsive factor [SRE], or NF-kB). Tax also affects NF-kB regulated transcription in a less direct manner. IkB binds to NF-kB dimers in the cytoplasm and prevents their nuclear localization. Inhibitor of kB kinases (IKKs) phosphorylate IkB causing it to dissociate. NF-kB can then migrate to the nucleus and activate transcription of cellular genes while IkB is degraded via the proteosome pathway. HTLV Tax is known to stimulate the activation of NF-kB at several levels in this pathway. Tax acts to free NF-kB from cytoplasmic constraints by causing its dissociation via the activation of IKKs and by acting as a molecular chaperone for proteolytic degradation due to its ability to physically associate with both the phos-phorylated form of IkB and the proteosome. By this mechanism, Tax would be able to guide IkB to the proteosome to promote its degradation and prevent further association with other cytoplasmic NF-kB dimers. Tax also enhances the trans-activating capabilities of NF-kB by stimulating its dimerization.

overall viral RNA transcription. Hence, it is the constant interplay of TAX and REX in concert with host cellular factors that influences the levels of HTLV proteins and virions being expressed at any given time and presumably influences the development of disease.

In vitro and in vivo studies indicate that HTLV RNA and proteins are expressed at much lower levels than HIV. This is explained in part by the more varied and robust repertoire of enhancer elements located in the HIV LTR. However, HTLV-infected cells have also been found to harbor natural anti-sense RNA molecules and to make predominantly multiply spliced rather than singly and unspliced viral mRNAs.40 These observed differences in infection and expression rates make teleological sense given the difference in biological activity of the two viruses. HTLV, which transforms its host cell, expands its genome by increasing the number of host cells containing integrated proviral DNA.43 Its low expression rate and high degree of latency decrease the probability of immunologic destruction. Hence, it has a low mutation rate.12 HIV, which ultimately destroys its host cell, requires much higher levels of infectivity, expression, and mutation in order to avoid immuno-logic inactivation and infect new host cells. The evolution of these biologic properties explains the different clinical effects (oncogenesis for HTLV, cytopenia for HIV) of these two T-lym-photropic viruses.

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