HTLV1 Replication Cycle
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HTLV1 Replication Cycle

The retroviral life cycle begins in the nucleus of an infected cell. At this stage of the life cycle the retroviral genome is a DNA element integrated into and covalently attached to the DNA of the host cell. Deltaretroviruses are complex viruses characterized by “C”-Type morphology. The most famous examples are the HTLV1 (Human T-Lymphotropic Virus-1), HTLV2 and the BLV (Bovine Leukemia Virus). HTLV1 is associated with a neurologic degenerative disorder known as TSP (Tropical Spastic Paraparesis) or, more commonly with HAM (HTLV1-Associated Myelopathy). HTLV1 spread occurs via semen, needles, breast milk and blood (Ref.1 & 2). There is no present licensed vaccine against HTLV1. Attachment of the virus occurs via SU (Surface Glycoprotein) peplomer. Attachment receptor for both HTLV1 and 2 is a cellular glucose transporter protein called GLUT1 (Glucose Transporter-1). These viruses penetrate via fusion, uncoat to core and then the Provirus is made through random integration. HTLV1 enters the body primarily through infected CD4+ lymphocytes. HTLV2 is found predominantly in CD8+ cells. HTLV1 carries no viral oncogene. The oncogenic potential of the virus is linked to the regulatory gene Tax (Transactivator X), where X is undefined. Retroviral Env (Envelope) glycoprotein mediates the attachment and subsequent membrane fusion of virions and infected cells with target cells. The native HTLV1 Env consists of a SU known as gp46 (Glycoprotein-46) and a non-covalently associated TM (Transmembrane Protein), gp21, formed by proteolytic cleavage of a polyprotein precursor, gp62. Receptor binding triggers conformational changes in retroviral SU-TM complexes that result in TM-mediated fusion. Exposure of glycoproteins to endosomal pH (pH 5-6) induces fusogenic activity. This fusion activation involves a helical extension of the central coiled-coil N-terminus to relocate the fusion peptide from the glycoprotein core to an envelope-distal location for insertion into the target cellular membrane (Ref.3 & 4).

The genetic structure of HTLV1 is similar, but distinct from other retroviruses. The viral genome is single-stranded, ssRNA of (+) polarity, 9-10 kb in length and diploid. Like eukaryotic mRNA it is capped at 5’ end and polyadenylated at 3’ end. There are 5 UTR (5 Untranslated Region) and 3 UTR (3 Untranslated Region). At the termini of the UTR’s an R (Repeat) sequence having 18-250 nucleotides is present (Ref.5). Internal to the R sequences there are non-repeated sequences U5 (Unique 5) and U3 (Unique 3) consisting of 75-250 nucleotides and 200-1200 nucleotides, respectively. The protein-encoding region lies between the UTR’s. It comprises sub-regions like gag, pro, pol and env, which are expressed as polyproteins. Polyproteins common to all retroviruses include Gag (Group Antigen), PR (Protease), Pol (Polymerase) and Env proteins. Gag polyprotein precursor is cleaved into three internal structural proteins like a 19 kd MA (Matrix) protein, a 24 kd CA (Capsid) protein and a 15 kd NC (Nucleocapsid) protein (Ref.6). In each group of retroviruses, these proteins are antigenically similar. Gag-Pro-Pol polyprotein contains, in addition to the internal structural proteins, the virion enzymes: PR, RT (Reverse Transcriptase) and IN (Integrase). Pol refers to the DNA polymerase activity of RT. Production of the Gag-Pro-Pol polyprotein requires a ‘frame shift’ by the translating ribosome. Gag-Pro-Pol polyproteins are assembled into virions as such and are not cleaved until the new virion buds from the plasma membrane. Cleavage is performed by the PR domain of the Gag-Pro-Pol polyprotein. This viral PR is the target of anti-viral drugs, if polyproteins are not cleaved, new virions does not become infective. The PR is encoded by pro gene (in the 3 part of the gag gene and the 5 part of the pol gene); synthesis of the PR is accomplished by ribosomal frameshifting. MA, CA and NC are encoded by the gag gene; RT and IN are encoded by the pol gene. Pol precursor is an 896-amino-acid protein. SU and TM are encoded by the env gene. Thus transcription and translation yields polyproteins like Gag, Gag-Pro, Gag-Pol and Gag-Pro-Pol that are cleaved by the viral protease or PR. But Env polyprotein precursor is cleaved by cellular protease (Ref.7 & 8).

All RTs basically fold conformationally like DNA Polymerase-I, with a folded palm like structure. The palm like region has sites for template binding and an active site for extension of the primer thus shows 5’->3’ polymerase activity. The connection between the palm and the thumb, called connecter has presumably the RNaseH (Ribonuclease-H) activity. Here the template used first is RNA and later DNA is used as the next template. Though it has no apparent 3’->5’ exonuclease activity, that means it lacks in proof reading function, yet mutation rate is not that high as expected of such enzymes. The RNaseH part of the enzyme is unique in the sense; it removes RNA only when it is hybridized to DNA. RNaseH means, it shows RNase activity only when it is hybridized to DNA. It cuts the RNA randomly and displaces the RNA fragments (Ref.6). It chews up RNA mostly from 5’ end and also from 3’ end. The enzyme has strong affinity to ssRNA and least affinity to dsDNA. The primer is degraded by RNAaseH. Because viral RNA is plus-sense, the first strand of DNA synthesized is referred to as (-) DNA. The (-) strand is extended to the end of the viral RNA, which is degraded by RNAaseH. The new (+) strand then “jumps” to the far end of the (-) strand, where it anneals via PBS (Primer Binding Site). Both DNA strands are then completed; RNAaseH destroys the remaining RNA. Regulatory sites in the viral genome includes-(i) E: A site required for Encapsidation of the genome. In the 5’ UTR; (ii) P (Promoter): Contains the transcription start site, as well as upstream sites for binding of cellular proteins which regulate transcription. Paradoxically, in U3, P is downstream from the genes instead of upstream. P is moved to an upstream position during reverse transcription; (iii) PBS: This 18 nucleotide sequence in viral RNA complementary to the primer which initiates reverse transcription (Ref.7). This primer is a cellular tRNA which is bound to the viral genome in the virion; (iv) Leader-It is a relatively long (90-500 nucleotides) non-translated region downstream of the transcription start site and therefore present at the 5 end of all virus mRNAs, (v) PPT (Polypurine Tract)-A short (approximately 10) run of A/G residues responsible for initiating (+) strand synthesis during reverse transcription; (vi) U3-A unique non-coding region of 200-1,200 nucleotides which forms the 5 end of the Provirus after reverse transcription; contains the Promoter elements responsible for transcription of the provirus. Apart from regulatory sites also include these SD (Splice Donor) and SA (Splice Acceptor). Splicing takes place between paired SD and SA sites. All retroviruses have at least one SD-SA pair, because Env protein is only be made from a spliced transcript (Ref.8).

HTLV1 genome also contains a region identified as pX, which lies 3 to the env sequence. This pX region is reported to contain five ORFs (Open Reading Frames), termed pX-I, pX-II, pX-III, pX-IV and pX-V. The pX region is highly conserved in HTLV1 and HTLV2 genomes. ORFs pX-I-V encodes the viral regulatory and cccessory proteins by alternative splicing of mRNAs. The main regulatory proteins are Tax and Rex (Regulator X), where X is undefined. The ORF III and IV encode for regulatory proteins, whereas, ORF I and II encodes for accessory proteins like p12, p12(I), p21, p21Rex, p27, p27(I), p27Rex, p13(I), p13/p13(II), p30, p30(II)/Tof, p40, Rof etc. These HTLV1 accessory proteins are critical for establishment of viral infectivity, enhance T-lymphocyte activation and potentially alter gene transcription and mitochondrial function (Ref.8 & 9). HTLV1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes. Function of ORF V is not known which means protein encoded by this region is not really detected. Tax transactivates many cONCs (Cellular Oncogenes). Tax protein co-localize primarily in nuclear bodies with RNA Polymerase-II , splicing complexes and specific transcription factors. Three imperfect 21-nucleotide repeats, termed TRE1 (Tax Response Element-1) are necessary for trans-acting transcriptional activation by Tax. U3 is responsible for termination and polyadenylation of mRNAs. Rex proteins occur in 27 or 21 KD form for HTLV1. Larger Rex protein is involved in the control of HTLV gene expression, but the function of the smaller HTLV protein (p21) is not clear. Rex regulates the nuclear exit of mRNAs encoding structural proteins and thus promotes virion production. Both Tax and Rex are essential for viral replication and cellular transformation. The overall (+) ssRNA genome of HTLV1 can be summarized as cap-R-U5-PBS-gag-pro-pol-env-pX Region-PPT-U3-R-polyA, where R-U5 is 5’UTR and U3-R is 3’UTR. The virion RNA and the Provirus differ in structure. The differences are created during reverse transcription. Virion RNA has R-U5 at the 5 terminus and U3-R at the 3 terminus (Ref.10). In the Provirus both termini contain the sequence U3-R-U5 which is otherwise known as LTR (Long Terminal Repeat). In virion RNA the “Promoter” lies within U3 at the ‘wrong’ end of the molecule. After the formation of the LTR, P is moved to an upstream position during reverse transcription. The Provirus genome can be summarized as U3-R-U5-PBS-gag-pro-pol-env-pX Region-PPT-U3-R-U5. In the retroviral DNA, the upstream LTR directs transcription. Another LTR directs the cleavage of the primary transcript to add a poly-A tail. Effects of Rex are mediated through specific cis-acting sequences in the LTR. Two cis-acting sequences: RxRE (Rex-Responsive Element), which allows Rex to overcome the inhibitory effect of a second, termed the CRS (Cis-Acting Repressive Sequence). The U5 in LTR contains sequences functionally defined as the CRS. Phosphorylated Rex localize in the nucleus (particularly in the nucleoli) (Ref.11).

IN searches the DNA for an appropriate “home”, whereupon the IN clips the host DNA and sews the double-stranded DNA into the host DNA. The virus is now prepared to initiate a new round of replication. Human pathogenic retroviruses do not have common loci of integration (Ref.1). However, many factors, such as chromatin structure, transcriptional activity, DNA-Protein interaction, CpG methylation and nucleotide composition of the target sequence, influence integration site selection. “CpG” stands for Cytosine and Guanine separated by a phosphate which links the two nucleotides together in DNA. The HTLV1 integrates in A/T-rich regions and these features are not associated with any preference for integration in transcriptionally active regions or in repeat elements of the host genome. Integration of Proviral DNA into the host genome generates short direct repeats of 4 to 6 base pairs as a result of DNA repair to the cellular sequence flanking the integrated Provirus. HTLV1 integration is not completely random in vivo. The structure of the target DNA plays an important role for integration in vivo (Ref.12).

Progeny Genomes and mRNA are produced by transcription. The enzyme responsible is host-cell RNA Polymerase II, the enzyme which carries out mRNA synthesis. The Promoter lies in U3 of the left-hand LTR and transcription starts at the first base of R. The polyA addition site is at the 3 border of R in the right-hand LTR. Thus transcription exactly re-creates the viral genome. A major unanswered question is why the Promoter sequence in the “downstream” right-hand U3 is not active. One hypothesis is that transcription complexes entering it from the right prevent binding of required transcription factors (“Promoter Occlusion”). Translated proteins assemble a retroviral particle at the cell surface. Full-length genomic unspliced mRNA (containing a packaging signal termed Psi) is bound by gag-derived proteins and incorporated into the budding particle. Env proteins are inserted into the plasma membrane. On the cytosolic side, they are bound by MA domain of uncleaved Gag (and a few Gag-Pol) polyproteins. Genomic RNA’s associate with the NC domain of Gag at this time. During budding, the PR domain of the Gag polyprotein cleaves Gag and Gag-Pol into their individual component proteins. This cleavage is essential for virion infectivity. Finally assembly of the HTLV1 occurs as “C”-Type particle at cell plasma membrane (Ref.4). HTLV1 was diagnosed in 1978 from a 28-year-old African-American with ATL (Adult T-Cell Leukemia/Lymphoma). HTLV2 was identified in a T-cell line established from a patient with atypical Hairy-Cell Leukemia in 1982. HTLV2 has a high nucleotide similarity to HTLV1. Both viruses have high stability and low mutation rate. HTLV3 was discovered in 1983 from patients suffering from an AIDS (Acquired Immune Deficiency Syndrome). The virus was later named HIV (Human Immunodeficiency Virus). HTLV1 has evolved/mutated very little even over thousands of years. Mummies excavated from the Atacama Desert in north Chile, which are estimated to be 1,300-1,700 years old, have ancient HTLV1 DNA that are almost identical to the viral DNA sequences of modern-day Chilean and Japanese HTLV1-seropositive individuals. In infected individuals, HTLVs show latency for years. Only precaution is the cure (Ref.13).