A unique feature of Retroviruses concerns their replication mechanism in host cells and organisms, a reverse transcription-integration process whereby the single-stranded viral RNA is copied by the viral DNA polymerase to generate the proviral DNA, which is integrated into the host cell DNA [1,2,3,4,5,6,7,8,9]. A genetic insertion such as this can impact on the life of the host and in some instances that of its descendants. In that respect, Retroviruses appear to be unique since no other infectious agent of higher eukaryotes is capable of integrating its genes into the host genome, of acquiring cellular genes into its own genome or has played such seminal roles in modern biology, biotechnology and in gene therapy [10,11,12,13].

Among Retroelements, the LTR-containing retrotransposons such as yeast retrotransposons (Ty) and human endogenous retroviruses (HERV) resemble simple retroviruses such as the gammaretrovirus murine leukemia virus (MuLV). In fact, these mobile elements encode the virion structural Gag proteins and Pol enzymes and contain non-coding regulatory sequences essential for genome replication, integration and expression such as the LTR (long terminal repeats). The endogenous retrotransposons that are abundant genetic elements in the host genetic make-up are probably playing key roles in genome reshuffling and variability. Thus, the replication of mobile retrotransposons by a transcription/reverse-transcription/integration process, also called ‘copy-and-paste’, is thought to have fueled the evolution of eukaryotic genomes from yeast to human (Figure 1 and legend) (reviewed in [14,15,16,17]).

Retroviral particles or virions have a globular structure with a mean diameter of 100 nanometers. The virion outer envelope is of cellular origin and contains the viral glycoproteins called surface (SU) and transmembrane (TM), in the form of trimeric ensembles for HIV-1. The shape and oligomeric organization of the structural proteins of the inner core seem to be characteristics for each genera of the Retroviridae family. The virion genomic RNA is 6,000 to 12,000 nucleotides in length with a positive polarity, and resides within the core as a 60S RNA complex, where two full length viral RNA molecules interact with each other and are coated by several hundred nucleocapsid protein molecules (about 1500 molecules in the case of HIV-1 and MuLV [18,19,20,21]).

Furthermore, it is important to briefly point out that actively replicating retroviral populations can have a complex composition, containing notably defective retroviruses which replicate only with the help of a replication competent retrovirus, called helper [4]. In fact, defective retroviruses commonly found in retroviral populations, need part or all of the functions of a fully competent retrovirus to replicate and disseminate in cells and organisms. Canonical defective retroviruses include highly oncogenic DLV’s (defective leukemia viruses) such as the Harvey and Kirsten MSV, which carries the v.ras oncogene flanked by retrotransposon VL30 sequences. The Moloney murine leukemia virus (MoMuLV) provides the viral helper functions in trans within the same cell to ensure co-replication of these DLV’s (see [22] for review).

The full length viral RNA, in addition to being the retroviral genome, plays a key role early in retrovirus assembly since it represents a scaffolding platform onto which Gag polyprotein molecules readily assemble to ultimately form the inner core structure [45-47]. In addition to the dimeric RNA genome, which is the reverse transcription template, the mature virion core structure contains subgenomic and a subset of cellular RNAs that can be reverse transcribed.

The overall process of Retrovirus assembly is considered to take place at the plasma membrane of infected cells where Gag and Gag-Pol molecules assemble via major interactions between two platforms, the N-terminal myristate and basic residues of the Gag-Matrix with plasma membrane phospholipids, and the zinc finger and the flanking basic residues of the Gag-NC with the genomic RNA (reviewed in [75,76,77,78,79]). Once completed, immature retroviral particles bud from the plasma membrane. Next, virion Gag and Pol molecules are processed by the viral protease (PR) during which condensation of the inner core occurs and virions gain infectivity [80,81]. However, retroviral assembly can also take place within infected cells, notably at the level of intracellular membranes such as endosomes and multivesicular bodies [82,83,84,85]. Under these circumstances the viral protease can readily remain active during assembly, directing the processing of Gag and Gag-Pol polyprotein precursors. Indeed, a large body of evidence obtained by western immunobloting demonstrates the presence of mature matrix, capsid and nucleocapsid proteins in cytoplasmic extracts of infected cells. Furthermore, the newly made virions contain a single copy of the full length viral RNA in a dimeric form along with minor quantities of spliced RNAs (see above) [49].

However, it has long been shown that small amounts of minus-strand viral DNA are present in viral particles of Rous sarcoma virus (RSV), Moloney murine leukemia virus (MoMuLV), and HIV-1 generated in cell cultures. This indicates that reverse transcription can already be ongoing during virus assembly or in the released particles, in a process which has been called natural endogenous reverse transcription (NERT) [86,87,88,89]. This early or premature reverse transcription has been extensively studied by the groups of Pomerantz and Mougel [50,90], revealing that AZT treatment of HIV-1 infected T cells causes a 10-100 fold reduction of intravirion viral DNA levels. This supports the notion that reverse transcription can take place, at least in part during the course of virus assembly as suggested some time ago in studies demonstrating that the synthesis of a complete infectious viral DNA can take place within virions of MoMuLV and equine infectious anemia virus (EIAV) under in vitro conditions using mild detergents that preserve the core integrity [92,93].More importantly, the physiological microenvironment, notably components of seminal fluid such as polyamines, dNTP’s and fragments of the abundant semen marker prostatic acidic phosphatase (PAP)s, can drastically enhance NERT and the accumulation of intravirion viral DNA [31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90]. HIV-1 virions containing high levels of viral DNA can readily infect non-dividing target cells. In addition, the PAP-derived peptide, termed Semen-derived Enhancer of Virus Infection (SEVI) that is also abundant in seminal fluid can efficiently promote attachment of HIV-1 particles to target cells. This in turn enhances viral infection and most probably facilitates the very early events of HIV-1 infection during sexual intercourse [31,90,91].

Furthermore, SEVI seems to display a potent nucleic acid chaperoning activity and to greatly enhance the reverse transcription process in vitro as well as in HIV-1 virions (Darlix JL et al., unpublished data), which suggests that SEVI might act as a multifunctional cofactor enhancing HIV-1 infection through numerous modes.

Taken together these findings show that viral DNA synthesis can indeed start during virus formation and that the so-called NERT process is largely influenced by components of the physiological microenvironment such as polyamines, dNTP’s and SEVI. This in turn can have a significant impact on HIV-1 infectivity of primary target cells [89,90,91].

Arguably, Retroviruses such as HIV should be viewed as RNA/DNA viruses rather than being strictly categorized as RNA viruses. Furthermore, the evidence highlights the need to identify microbicide compounds aimed at inhibiting components of the seminal fluid which facilitate HIV-1 infection [94].

In the course of assembly, Gag structural precursor molecules are targeted to the plasma membrane by the N-myristate and basic residues of the Gag matrix (MA) on the one hand, and the genomic RNA, notably the Psi Packaging signal (its stem-loops specifically bound by the C-terminal Gag nucleocapsid (NC)), on the other. In fact, NC – consisting of either one or two highly conserved zinc fingers flanked by basic residues – directs the selection of the genomic RNA through multiple interactions between the RNA Psi sequences and both its zinc finger(s) and its basic residues [20,21,75,95].

In the case of HIV-1 NC, the central globular domain corresponding to the two zinc fingers and the basic linker forms a hydrophobic platform which specifically binds the Psi stem-loops of the genomic RNA (reviewed in [21,75]). Mutating the highly conserved CCHC residues of the zinc fingers causes an impairment of genomic RNA packaging in newly formed virions. Interestingly, these CCHC mutations result in profound modifications of Gag trafficking in cells and in the production of viral particles that are completely replication defective (reviewed in [20,95]).

However reverse transcription does take place in CCHC mutant virions as evidenced by the accumulation of newly made viral DNA ([96,97], and ref. herein), which does not, however undergo integration. The observed reverse transcription occurs during assembly and prior to virion budding because addition of the nucleoside RT inhibitor (NRTi) AZT to the producer cells blocks accumulation of the viral DNA. This newly synthesized viral DNA, or at least part of it, was found to be functional since it was capable of promoting synthesis of the viral Tat transactivator and of LTR activation ex vivo [96].

The above data highlight the fundamental role of NC in virus assembly and reverse transcription, and possibly in the maintenance of the newly made viral DNA [40] (reviewed in [20,95]). In addition, there seems to be a tight connection between virus assembly and the start of reverse transcription where slowing down or modifying the assembly process would be expected to modify the timing of viral DNA synthesis. The data also favor the notion that minor structural modifications of the inner virion core would result in the intracellular instability of newly made viral DNA, notably at the 5’ and 3’ ends. Indeed, mutating the NC zinc fingers results in the formation of HIV-1 particles with a general globular structure and not a condensed cone-shaped structure as typified by the wild type virus [99], which result in degradation of the incoming virion core by cellular factors (see § on the synthesis of functional viral particles in primary cells, below).

In support of such a dynamic connection between virus assembly and budding, and viral DNA synthesis, Thomas et al. [98] reported that mutating the p6 PTAP motif within the Gag C-terminal domain slowed virion budding, an effect probably due to weakening of the interaction between Gag and the cellular transporter protein TSG101. At the same time, this causes extensive premature reverse transcription and the accumulation of newly made viral DNA in p6 mutant virions.

Thus, the general view that emerges suggests that the dynamics of the assembly-budding process exert tight control on the timing of viral DNA synthesis by RT, chaperoned by NC in the virion core. Furthermore, the global structure of the core, central to which the multiple interactions between NC molecules and the genomic RNA, would transiently function as a shield against cellular factors capable of degrading components of the viral core, which would otherwise prevent synthesis of a functional viral DNA. For highly replicating viruses such as HIV-1, the nature of the molecular interactions between the genomic RNA, tRNA, RT and NC, and between the viral DNA, NC and IN largely out competes the influence of host restriction factors such as APOBEC and TRIM proteins. In addition, these tight molecular interactions might well be indispensable for the synthesis of complete viral DNA containing mutations and reassortment of large regions by means of recombination reactions. This should confer, at least in part, resistance to immune responses and anti-retroviral therapies targeting the RT, PR and IN enzymes (HAART).

In cells undergoing HIV-1 infection, the process of reverse transcription occurs in a densely populated environment, the cytoplasm. There, it is intimately linked to at least two other steps, namely uncoating, the process through which the viral core is reorganized, and trafficking towards the nucleus [100]. Given that reverse transcription occurs in this viral shell that serves several functions, it is not surprising that this process is intimately linked to the fate of viral cores. Indeed, mutations that affect the core major structural component, (i.e. the Capsid protein (CA)), almost invariably display a defect in viral DNA accumulation [100,101,102,103].

However, the relationship between HIV-1 reverse transcription and viral core reorganization is poorly understood. Specifically, it is not known whether uncoating occurs subsequent to the completion of viral DNA or if a reciprocal co-stimulation is established between the two processes. The former hypothesis was raised following evidence indicating that viral cores gained stability in the absence of the central polypurine tract -central termination sequence (cPPT-CTS) cis-element [104]. In this debated model, the three-stranded DNA flap formed in the neo-synthesized viral DNA, conveys the signal of end of reverse transcription which starts viral core restructuration and prepares it for the next step of the viral life cycle, i.e. nuclear import. This model is seducing as it finds parallels in other viruses whose capsids undergo profound changes in the proximity of nuclear pores that are required for the nuclear import of their genome [104,105,106]. Whether this is true for HIV-1 remains a matter of debate as biochemical evidence suggests that extensive viral core reorganization occurs either prior to or during reverse transcription [107,108,109,110] and cPPT-CTS mutants display rather moderate infectivity defects [111,112,113].

Why is the relationship between reverse transcription and uncoating important? We propose it is so to counteract in a timely fashion possible antiviral defenses that target viral cores. Viral cores are characteristic molecular signatures of Retroviruses and they are known to be targeted shortly after cell entry by antiviral factors. For example, the Tripartite motif protein 5 alpha (TRIM 5α) recognizes viral cores and destructures them, impacting on reverse transcription (108). If uncoating does not occur until the completion of viral DNA synthesis, then a structure identical to the one entering the cell will be present throughout the journey of the viral core across cytoplasm. If, instead, uncoating occurs progressively together with reverse transcription this characteristic complex will undergo change, as a consequence of which the viral structures sensed by cellular antiviral defenses will be markedly distinct.

It remains to be seen if TRIM5α or the Friend susceptibility virus gene 1 (Fv1) are unique factors or if other proteins exert similar functions, but it would not be surprising to find these characteristic retroviral structures to be the target of multiple factors. In this respect, by providing a signal for the ordered reorganization of viral cores, reverse transcription may protect the virus from attack within the cytoplasm environment simply by promoting shedding of the components through which viral cores are recognized.

The major difference between the two models outlined above on the relationship between core reorganization and reverse transcription is the length of time during which the initial viral core structure persists as such in the cytoplasm of infected cells. It is well appreciated that viral DNA completion varies greatly among target cells. For example, while the reverse transcription process takes only 4 to 8 hours in transformed cell lines such as HeLa cells, widely used to study viral infection, it can take up to 20-30 hrs in primary lymphocytes and macrophages or days in non-stimulated monocytes [114,115,116]. Thus, the extent of reverse transcription in different target cells is extremely variable and dependent on the activation status of target cells, the dNTP pool and on the plethora of cellular factors that are present in these cells at the moment of infection. In cells in which reverse transcription occurs slowly, antiviral defenses are more likely to recognize and counteract incoming virion cores.

Indeed a correlation between the extent of reverse transcription and the permissivity of primary cells to infection is widely reported. In the past, this has been hypothesized to be a consequence of poor availability of dNTPs in cells resistant to infection. Yet, more recent data indicates that RT functions correctly even at low dNTP concentrations, such as those normally found in restrictive cells including macrophages or monocytes [116,117]. It is thus conceivable that the correlation between the rate of reverse transcription and a cell’s susceptibility to infection is due to a protective effect of the viral core permitting more efficient reverse transcription and thus infection.

One of the major problems in studying the early phases of infection is the long appreciated observation that the vast majority of viral particles entering cells are non-infectious, as they do not result in the establishment of an integrated proviral DNA and are progressively lost over time. Only 1 in 8 viruses is thought to be infectious in established cell lines where infection is efficient [118] but may well be at least 1 in 100 particles in primary cell types such as macrophages and dendritic cells. The reasons for this are unclear at present and defects at the level of viral assembly cannot be excluded. Yet, an interesting hypothesis is that intracellular defenses are largely responsible for the generation of non-infectious viruses, in which replication terminates at some point despite successful target cell entry and initiation of reverse transcription. This hypothesis remains unaddressed.

Studying reverse transcription in HIV-1 has shed some light on virus variability fueled by the error prone RT enzyme, which is, at least in part, counterbalanced by NC protein in order to control the process and to contribute to its completion and fidelity (Figure 4). At the same time, NC and NC-RNA interactions most probably drive the incorporation of cellular deaminase APOBEC 3G, which should in turn contribute to HIV-1 variability (Figure 4), though it in turn counterbalanced by the viral factor VIF.

To understand how HIV-1 genetic variability allows the virus to resist innate defenses, specific immunological responses and highly active antiretroviral therapies targeting the viral enzymes RT, PR and IN (HAART) [119], the circulating virus populations should be viewed as quasi-species consisting of genetically distinct but closely related viruses. Analysis of HIV-1 proviral DNA in individual T-cells present in the lymph nodes of infected persons shows a substantial fraction harbor distinct proviruses [120]. It follows that infected T-cells possess the ability to produce both homozygous and heterozygous viruses by means of recombination during reverse transcription, further fueling HIV-1 variability [18,21,121,122] and translates into the generation of a large number of virus quasi-species.

Since the nucleocapsid protein of HIV-1 plays key roles in reverse transcription, mainly through its highly conserved zinc fingers, it represents a target of choice for compounds and approaches [123] that seek to complement HAART and impair the circulation of HIV-1 strains resistant to anti-RT, -PR and –IN drugs [120].