Which viruses have reverse transcriptase

The series of reactions carried out by RT to copy the retroviral genome in order to generate the double stranded viral DNA which is then integrated into the host-cell genome has been known for almost 25 years.

The dimeric nature of the retroviral RNA genome is largely responsible for the high genetic variability of highly replicating viruses such as Rous sarcoma virus RSV and HIV-1 by means of forced and non-forced copy-choice recombinations during reverse transcription [ 12 , 14 , 15 ]. A scheme of the HIV-1 replication complex. The genomic RNA in a dimeric form is coated by about nucleocapsid NC protein molecules in red in the viral core particle. For references see text. As a result, the structure-function relationships of HIV-1 RT have been and continue to be intensively studied using a multidisciplinary approach.

The 3D structure of RT in its pp51 free form was established [ 15 — 17 ], and more recently the specific orientation of the RT polymerase and RNaseH active sites was characterized using single molecule assays in vitro [ 18 , 19 ].

Later, it was discovered that the major virion protein of the inner core of alpha and gamma-retroviruses and lentiviruses, the nucleocapsid protein NC encoded by Gag was a key cofactor of the RT enzyme, chaperoning obligatory steps in viral DNA synthesis [ 20 — 28 ]. At the same time, the NC domain of the Gag structural polyprotein was found to direct genomic RNA selection, packaging and dimerization during virion assembly [ 29 — 34 ].

Thus NC is a multifunctional virus structural protein necessary for the completion of the early and late phases of retrovirus replication reviewed in [ 28 , 35 — 38 ].

How then can we explain the multiple roles of NC? NC is a potent nucleic acid chaperone, which tightly binds nucleic acids and facilitates the annealing of complementary sequences as well as strand transfer and exchange reactions in physiological conditions reviewed in [ 35 — 38 ]. NC is encoded by Gag in most, if not all, retroviruses and retrotransposons [ 36 ] where its unique chaperoning activity ensures primer tRNA annealing to the genomic PBS and the obligatory minus and plus DNA strand transfers that are required for the synthesis of the complete, LTR flanked, viral DNA [ 28 , 37 , 38 ].

These multiple RT-NC-RNA interactions contribute to the fidelity of the reverse transcripton reaction by inhibiting self-initiation of cDNA synthesis and providing excision-repair activities to the RT enzyme in vitro [ 24 , 39 , 41 — 46 ]. After cell infection, the virion core is released into the cytoplasm where it is believed to undergo structural alterations giving rise to a large ribonucleoprotein structure called the reverse transcription complex RTC , the site of extensive viral DNA synthesis.

A different view was recently provided by biochemical and electron microscopy studies showing that HIV-1 cores remained in the cytoplasm of newly infected cells up to the nuclear pore [ 50 — 54 ]. These results strongly suggest that completion of proviral DNA synthesis most probably relies on the proper structure and the stability of the viral cores. The canonical view of retrovirus formation, notably that of HIV-1, states that the overall process takes place at the plasma membrane where Gag molecules assemble via interactions between MA and the phospholipids on the one hand, and between NC and the genomic RNA on the other reviewed in [ 55 ].

Upon completion, immature particles are produced by budding during which Gag and Pol processing by the viral protease PR occurs, ultimately leading to the condensation of the inner core [ 32 , 56 ]. However, a series of results indicate that assembly can also take place on intracellular membranes such as endosomes and multivesicular bodies [ 57 — 60 ].

The PR enzyme may therefore already be active at the onset of assembly directing the cleavage of the Gag and Gag-Pol polyproteins, as evidenced by the presence of mature CA, MA and NC proteins in cytoplasmic extracts of infected cells. In both cases, the newly made viral particles are thought to contain the full length viral RNA in a dimeric form as the genetic material along with minor quantities of spliced RNAs [ 61 ]. This notion of premature reverse transcription has been confirmed by Zhang et al.

In addition, the physiological microenvironment, for example the seminal fluid, was found to enhance the accumulation of intravirion viral DNA by a process called natural endogenous reverse transcription NERT [ 64 ].

Interestingly, synthesis of a full length infectious viral DNA can be achieved in virions of MLV and equine infectious anemia virus EIAV under well defined in vitro conditions [ 6 , 65 ], that probably reconstitute the microenvironment promoting extensive NERT, especially components present in the seminal fluid [ 66 ].

The newly synthesized viral DNA present in infecting virions was shown to play a key role in vivo because it augments virus infection of non-activated human primary target cells by nearly one hundred fold while it has no effect on activated T cells [ 66 ]. The role of the physiological microenvironment is not limited to viral DNA synthesis since a recently identified aggregating prostatic acidic phosphatase PAP -derived peptide that is abundant in the seminal fluid was shown to augment virus to cell attachment and entry, thus facilitating the very early event of HIV-1 infection during a sexual intercourse [ 67 , 68 ].

It has long been shown that mutating the highly conserved CCHC residues of the NC zinc fingers impairs genomic RNA packaging and results in the production of replication defective viral particles reviewed in [ 28 , 37 , 38 ].

Moreover, mutations affecting the 3D structure of the zinc fingers or their respective orientation cause a decrease in the genomic RNA content of HIV-1 viral particles and result in the production of defective particles, although significant amounts of viral DNA can be synthesized in infected cells [ 26 , 27 , 45 , 69 ]; reviewed in [ 37 , 38 , 70 ]. However, this has been recently disputed. In fact, the influence of NC mutations on unsuspected aspects of HIV-1 virion formation has just been discovered, in particular the influence on packaging of multispliced 1.

It was found that deleting or mutating the NC zinc fingers or the N-terminal basic residues caused a 10—30 fold reduction of genomic RNA in newly made virions Fig. Gorelick and colleagues [ 74 ]. This reverse transcription takes place in virus producer cells since the addition of the RT inhibitor AZT prevented accumulation of viral DNA in virions, in agreement with the earlier findings of Pomerantz et al.

These findings not only confirm the key role of NC in RT-directed viral DNA synthesis and probably its maintenance [ 43 , 45 ]; reviewed in [ 70 ] but also indicate that NC exerts a control on the timing of reverse transcription.

How then can we explain this extensive reverse transcription already in cells producing the HIV-1 NC mutant virions? Within HIV-1 virions with mutations in or deletion of the NC CCHC zinc fingers, the core is formed of mature Gag proteins but it is mostly globular and does not adopt a condensed cone-shaped structure as seen by electron microscopy [ 75 ].

These results favor the notion that these NC mutations cause a defect in the late step of Gag assembly. Such ZF mutations also have a negative impact on NC-RT interactions in vitro [ 42 , 77 ] while the chaperoning activities of the NC mutant proteins either are not or are slightly affected [ 78 ]; reviewed in [ 37 ]. The study of Gorelick et al. Taken together, these findings support the notion that the highly conserved CCHC zinc fingers of NC control formation of a dense core structure where reverse transcription is prevented, at least, partially [ 72 — 74 ].

Thus, the viral NC protein would exert a control on the timing of viral DNA synthesis by the active RT enzyme, delaying the start phase in virus producer cells and chaperoning the entire process until completion in newly infected cells Figure 3. There are also indications that the conserved CCHC zinc fingers of NC contribute to the maintenance of the complete viral DNA and its IN-mediated integration into the host genome during the course of cell infection [ 45 , 79 , 80 ]. These processes are facilitated by interactions between NC and cellular proteins large black arrows.

The core containing the genomic RNA is condensed with a cone-shaped structure. They result in a partial delocalization of Gag in producer cells and a reduced level of newly made viral particles grey arrow see text for references. RTs of retroviruses and LTR retrotransposons are active as monomers e. Ty3 RT or heterodimers e. RTs lack proofreading activity and display high intrinsic error rates.

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