Macaque-Tropic HIV-1 Derivatives: A Novel Experimental Approach to Understand Viral Replication and Evolution In Vivo

The use of animal models in the study of human diseases is obviously important. Fundamental properties of the disease can be investigated analytically and thoroughly by this approach, contributing much to the progress of basic science as well as clinical medicine (Nomaguchi & Adachi, 2010). Researchers in various specialties, therefore, have made every effort to establish animal models for human diseases including those caused by infectious agents. Acquired immunodeficiency syndrome (AIDS) of humans has long been one of the major targets for the model study in appropriate animals. However, human immunodeficiency virus type 1 (HIV-1) itself, the predominant causative virus of human AIDS, can not be used due to its very narrow host range. Because HIV-1 has adapted itself dexterously from the ancestral virus to replicate, persist and spread strictly in humans, it is very unique among various primate immunodeficiency viruses and no good counterparts are available in nature (Desrosiers, 2007; Kirchhoff, 2009; Sauter et al., 2009). Therefore, it can be concluded that practical and meaningful animal systems of non-alternative nature for HIV-1 study do not exist at all to date, although there are pre-existing animal models of some significance. HIV-1 does not replicate in animal species except for chimpanzees and humans (Nomaguchi et al., 2008a). Animals frequently used for our experiments on virology, such as rodents and nonhuman primates, are not exceptions to this barrier. However, if we are to search for, develop and establish a fruitful animal model system for HIV-1 research, nonhuman primates are considered to be most suited, for HIV-1 is best fitted with humans and some apes. Ever since the discovery of HIV-1 (Barre-Sinoussi et al., 1983), many prominent researchers keen on understanding its biology and molecular biology have done investigations extensively to elucidate the bases underlying the species-specificity unique to HIV-1. These studies have highlighted the presence of potent anti-HIV-1 factors in nonhuman cells that efficiently restrict or even abolish the replication of HIV-1 and successfully raised an epoch-making notion of the intrinsic immunity (Andrew & Strebel, 2010; Arhel & Kirchhoff, 2010; Ayinde et al., 2010; Bergamaschi & Pancino, 2010; Douglas et al., 2010; Fujita et al., 2010; Huthoff & Towers, 2008; Kirchhoff, 2010; Luban, 2007; Malim & Emerman, 2008; Nakayama & Shioda, 2010; Nomaguchi et al., 2008a, 2008b; Planelles & Barker, 2010; Sauter et al., 2010; Strebel et al., 2009; Towers, 2007). Cellular factors shoulder


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this intrinsic immunity known to date are cyclophilin A (CypA) (Franke et al., 1994;Thali et al., 1994), apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G)/APOBEC3F (Sheehy et al., 2002), tripartite motif protein 5 (TRIM5) (Stremlau et al., 2004), TRIMCyp (Nisole et al., 2004;Sayah et al., 2004), and tetherin (alternatively called BST-2) (Neil et al., 2008;Van Damme et al., 2008). Because HIV-1 can indeed counteract human orthologs of these restriction factors effectively, it is well anticipated that HIV-1 in turn can be genetically engineered to replicate efficiently in nonhuman primates such as macaques. Nonetheless, most likely due to the lack of extensive and appropriate biological studies, we are still forced to use macaque-derived simian immunodeficiency virus (SIVmac) or SIVmac chimeric with a small portion of HIV-1 (SHIV) as an input virus for in vivo model studies in macaques. SIVmac and SHIV are genetically and biologically distinct from HIV-1 in a number of critical points, albeit they are quite similar to HIV-1 in the genome organization and pathogenic potentials (Desrosiers, 2007;Freed & Martin, 2007). They might not be used for future model studies aimed at understanding the biology of HIV-1 as a highly replicable/mutable, persistent, and pathogenic virus. We must go behind the outward form to grasp the inner meaning of the phenomenon, i.e., the species-specificity. On the collective basis of molecular and biochemical studies performed by us and others so far, we recently have constructed a series of HIV-1 derivative clones tropic for macaque cells and/or macaques (Hatcho et al., 2008;Igarashi et al., 2007;Kamada et al., 2006Kamada et al., , 2009Kuroishi et al., 2009;Nomaguchi et al., 2008a;Saito et al., 2011;Yamashita et al., 2008), and are currently further modifying them for in vivo studies (our unpublished results). The viruses we have generated carry a minimal sequence of SIVmac, and overcome at least some species barriers. Importantly, these viruses are regarded to be genetically HIV-1, since they have less than 10% SIVmac genetic content . While we firmly believe that HIV-1 derivative viruses already constructed in our laboratory are useful for a variety of studies on HIV-1 infection in individuals, further improvement of the viruses by deliberating the evolutional process of SIV/HIV would surely add more scientific significance to basic and applied research fields. Needless to say, our goal is to generate a macaque-tropic HIV-1 (HIV-1mt) that replicates efficiently and is pathogenic for macaques as a standard pathogenic SIVmac clone such as SIVmac239 (Kestler et al., 1990). Through construction and biochemical/biological characterization of the ideal HIV-1mt clone with ability to induce AIDS at least in some species of macaques, we would be able to clarify the detailed molecular mechanisms for the narrow host range (species-tropism) of HIV-1. Viral Gag-capsid (CA) and accessory proteins (Vif, Vpx, Vpr, Vpu and Nef) are targets for those studies as a matter of course. Moreover, by using this persistent and pathogenic HIV-1mt clone as a seed virus for macaque infection experiments, we can trace and analyze its mutation, adaptation, evolutional direction to generate viral quasi-species, and finally pathogenesis in the context of immunological interaction. In addition, we can evaluate and develop the anti-HIV-1 drugs/vaccines by this HIV-1mt/macaque system. In this chapter, we first outline the early and current studies on HIV-1, SIVmac and SHIV to emphasize and address the unique characteristics of HIV-1 and scientific issues to resolve. We then describe viral and cellular factors that are responsible for or potentially associated with restriction of viral replication. We finally focus on our recent studies on the strategies to obtain HIV-1mt clones and on the biology/molecular biology of HIV-1mt clones. Main parts of this chapter consist of: (i) Overview of the biology and molecular biology of HIV-1, SIVmac and SHIV; (ii) Determinants for HIV-1 species-tropism; (iii) Generation and 328 proteins are essential or important for virus replication. These findings have led to the identification of innate anti-viral factors APOBEC proteins (Sheehy et al., 2002) and tetherin as described above (Neil et al., 2008;Van Damme et al., 2008), and to the search for an antiviral macrophage factor(s) . Although some aspects of the accessory proteins are becoming more organized and much clearer than before as summarized in Table 1, detailed mechanisms for their activity remain to be elucidated. In particular, much is still unknown about structurally related Vpr and Vpx proteins. Moreover, functional studies in animals on HIV-1 and HIV-2 accessory proteins have not yet been performed.   (Freed & Martin, 2007 One of the most outstanding biological properties of HIV-1 is its especially narrow host range. It was recognized soon after the virus isolation that HIV-1 can not infect macaque cells and macaques, animals frequently used for experimental infection. We, therefore, pioneered the work to determine viral determinants for this species-tropism by construction and characterization of chimeric viruses between SIVmac and HIV-1 Sakuragi et al., 1992;Shibata et al., 1991Shibata et al., , 1995. SIVmac has a wider host range relative to HIV-1, and can efficiently replicate both in macaque and human cells. The chimeric viruses ( Fig. 4), later called SHIV, were useful to localize the viral genetic area responsible for the tropism. Among NM-1, NM-3, and NM-8 in Fig.4, only NM-3 was shown to display infectivity to macaque cells. In addition, Gag-CA region was suggested to be important for the tropism by a similar analysis of chimeric viruses (Dorfman & Gottlinger, 1996). Totally, these SHIV studies revealed that Gag-CA plus some viral protein(s) encoded by the central viral genomic region may determine the HIV-1 speciestropism.
As for input viruses of model infection studies in macaques, SIVmac and SHIVs have been widely and frequently used (Ambrose et al., 2007;. SIVmac is thought to emerge by a cross-species infection of rhesus macaques with SIVsmm naturally occurring in African sooty mangabeys (Fultz et al., 1986;Murphey-Corb et al., 1986). It targets CD4-positive cells such as T-lymphocytes and macrophages, persists, and finally cause AIDS in rhesus macaques. Pathogenic SHIVs have been obtained from the original prototype SHIV by serial animal passages, and were used for infection experiments in rhesus macaques. An SIVmac derivative that has reverse transcriptase (RT) of HIV-1 (RT-SHIV) (Fig. 4) was also constructed to test the effect of anti-RT drugs on virus replication (Uberla et al., 1995). Although these viruses did contribute much to HIV-1 model studies including the assessment of immune response, evaluation of anti-viral drugs, analysis of drug-resistance, and establishing the strategy for vaccine development, there are some intrinsic differences among important virological properties of HIV-1, SIVmac and SHIVs as summalized in Table 2. These should be seriously considered for the future model studies.
To underscore the essential need for the suitable primate model research to answer basic questions about HIV-1 in vivo, we wish to mention here, as an example, that the trials to develop anti-viral vaccines have been unsuccessful due to the lack of appropriate models (Hayden, 2008;Watkins et al., 2008).  (Adachi et al., 1986) and SIVmac MA239 (Shibata et al., 1991), respectively. Areas without gene names indicate that the genes there are inactivated by genetic manipulations.

Determinants for HIV-1 species-tropism
Our early studies on systematic analysis of HIV-1 proviral mutants by site-directed mutagenesis have clearly demonstrated the cell-dependent functionality of some viral proteins (Gag-CA, Vif, Vpu, and Vpx) and the cell-dependent viral replication (Adachi et al., 1999;Kawamura et al., 1994bKawamura et al., , 1998Sakai et al., 1993Sakai et al., , 1995Sakuragi et al., 1995). These results have strongly suggested the presence of specific intracellular factors, other than receptor molecules for viruses, responsible for viral cellular tropism. Importantly, restriction factors against HIV-1 (APOBEC3/Vif, TRIM5/Gag-CA, and tetherin/Vpu) have been recently identified and molecularly cloned (Neil et al., 2008;Sheehy et al., 2002;Stremlau et al., 2004;Van Damme et al., 2008). Furthermore, a new restriction factor functional in macrophages and antagonized by Vpx has been proposed Fujita et al., 2010;Sharova et al., 2008;Srivastava et al., 2008). Taken altogether, these findings have prompted active researchers to examine whether these cellular proteins are associated with the HIV-1 species-tropism. As results of a series of comparative biological and biochemical studies on the interaction between HIV/SIV and human/monkey restriction factors, it has been revealed that various species-specific cellular proteins in Table 3 determine or modulate the species-tropism of HIV-1. As can be understood in Table 3, viral accessory proteins Vif, Vpu, Vpx (and/or Vpr), and Nef (in the case of some SIVs) play significant roles (Tables 1 and 2 Table 3. Restriction factors against HIV-1. Cellular anti-HIV-1 factors identified and one of potential anti-viral factors are listed. As for the details of restriction factors of these two categories, see the text.
2008). It is well-predicted that primate immunodeficiency viruses now have evolved by acquiring the appropriate accessory genes through numerous mutations and recombinations (Kirchhoff, 2009(Kirchhoff, , 2010Sauter et al., 2009Sauter et al., , 2010. Among viral structural proteins, only Gag-CA, which constitutes a major virion component, appears to be deeply involved in the species-tropism of HIV-1. By adapting Gag-CA and accessory proteins to the hostile environment, HIV/SIV could spread, persist, and survive. In this regard, HIV-1 has developed its specific characteristics from the progenitor form, and may be still uniquely altering its virological property through multiple rounds of the infection cycle in human populations.

Vif and APOBEC3G/F
Accessory protein Vif (Table 1) is essential for HIV/SIV replication in certain cell types such as natural target cells (T-lymphocytes and macrophages) that express APOBEC3G/APOBEC3F. APOBEC3G/F are members of a polynucleotide cytidine deaminase family that displays www.intechopen.com Macaque-Tropic HIV-1 Derivatives: A Novel Experimental Approach to Understand Viral Replication and Evolution In Vivo 335 diverse functions (Holmes et al., 2007), and are potent inhibitors of viral replication counteracted by Vif. Vif degrades APOBEC3G/F via the ubiquitin-proteasome pathway (Table  1 and Fig. 3). In the absence of Vif, APOBEC3G/F are incorporated into virions, and cause lethal mutations in viral genome during the reverse transcription process in a new infection cycle (Table 3). There are two functional domains in Vif, that is, N-terminal binding region to APOBEC proteins and C-terminal region for degradation (Strebel et al., 2009). Noteworthy, HIV-1 Vif does not degrade APOBEC3Gs of the rhesus macaque and African green monkey probably due to its inability to binding to them. In contrast, SIVmac Vif can inactivate both human and simian APOBEC3Gs. Thus, the interaction of Vif and APOBEC3G/F is critically important for the unique species-tropism of HIV-1. In our experience, APOBEC3G/F is the strongest determinant for this tropism among the restriction factors listed in Table 3. Whether another activity of Vif to induce G2 cell cycle arrest (Izumi, T., 2010) (Table 1) is involved in the species-tropism is presently unknown.

Gag-CA and its interacting cellular proteins (CypA, TRIM5 and TRIMCyp)
Early studies have already indicated that Gag-CA is responsible for the HIV-1 speciestropism as described above (Shibata et al., 1991;Dorfman & Gottlinger, 1996). Recent works have focused on the interaction of Gag-CA and its counterpart (CypA, TRIM5 and a TRIM5/CypA fusion protein, TRIMCyp). It is well-established now that CypA, TRIM5 and TRIMCyp act as an inhibitor of HIV-1 replication in a species-specific manner (Lim et al., 2010;Luban, 2007;Nakayama & Shioda, 2010;Price et al., 2009;Towers, 2007;Ylinen, 2010). These cellular proteins exert their anti-viral powers on the incoming virion core in a poorly defined way (Table 3 and Fig. 2). Of note, CypA positively and negatively regulates HIV-1 replication in human and macaque cells, respectively. Importantly, rhesus TRIM5, cynomolgus TRIM5 and cynomolgus TRIMCyp effectively inhibit HIV-1 replication, but not rhesus TRIMCyp. Therefore, CypA, TRIM5 and TRIMCyp can determine the unique species-tropism of HIV-1. We estimate that Gag-CA is the second strongest determinant for the tropism. It should be stressed here that the polymorphism observed in TRIM5 alleles affects the sensitivity of hosts to virus infection.

Vpu and tetherin
Accessory protein Vpu (Table 1) is required for optimal replication of HIV-1 in certain cell types that express tetherin. Tetherin specifically inhibits the virion release from cells (Table  3) and is countered by Vpu (Nomaguchi et al. 2008b;Strebel et al., 2009). Vpu degrades cellular tetherin and CD4 effectively. It is generally accepted that Vpu enhances virion release from the cell surface by down-regulation of tetherin (Table1, Table 2 and Fig.3), and thereby promote viral replication. However, Vpu proteins of HIV-1 and some SIVs can not efficiently antagonize simian tetherin molecules relative to those of SIVs with a high ability (Sauter et al, 2009). In fact, HIV-1 NL4-3 scarcely suppressed the anti-viral activity of the rhesus tetherin. Based on this finding, it can be concluded that tetherin is associated with the species-tropism of HIV-1. However, in our experience, the positive effect of Vpu on viral replication is much smaller than those of Vif and Gag-CA. Moreover, another functional activity of Vpu to degrade cellular CD4 is considered to be irrelevant to the HIV-1 speciestropism. Whether Vpu is associated with the HIV-1 pathogenesis is an important question to address. Interestingly and importantly, Env of some HIV-2 isolates and Nef of some SIVs have the Vpu-like ability to enhance virion release (Strebel et al., 2009;Zhang et al., 2009).

Potential determinants for HIV-1 species-tropism
It has been recently reported that HIV-2/SIVmac Vpx is necessary for the post-entry step of viral replication, such as uncoating/reverse transcription, in monocyte-derived dendritic cells and macrophages Goujon et al., 2007;Srivastava et al., 2008). Vpx is supposed to counter an unidentified anti-retroviral factor(s) present in cells of this lineage (Tables 1-3 and Fig. 2). Because Vpx can also up-regulate the HIV-1 replication, the unidentified macrophage factor appears to be commonly important for HIV/SIV replication.
To substantiate the macrophage entity as a restriction factor against HIV/SIV and/or the other retroviruses, its identification is urgently required.
During a systemic characterization of HIV-1mt CA mutants, we have noticed a TRIM5independent enhancement of viral infectivity in macaque cells (Nomaguchi et al., manuscript in preparation). This result suggests the presence of unknown anti-viral factor that interact with HIV-1 Gag-CA. We also have found a mutation in the Env-SU region that confers the mutant a significant affinity to macaque CD4, considerably promoting virus replication (Nomaguchi et al., manuscript in preparation). These observations may be relevant to the HIV-1 species-tropism.

Generation and characterization of various HIV-1mt clones
To obtain a novel class of HIV-1 that infects, replicates and finally causes AIDS in macaques, we and a research group in USA have independently initiated the work on HIV-1mt and have done macaque model studies (Hatcho et al., 2008;Hatziioannou, 2006Kamada et al., 2006Kamada et al., , 2009Kuroishi et al., 2009;Nomaguchi et al., 2008a;Saito et al., 2011;Yamashita et al., 2008). Another group has published a report on HIV-1mt derivatives very recently (Thippeshappa et al., 2011). We now are actively and thoroughly amending the HIV-1mt genome by computer-assisted and structure-guided mutagenesis. Our prototype HIV-1mt designated NL-DT5R (Kamada et al., 2006) contains a 21-nucleotide SIVmac Gag-CA element (corresponding to the HIV-1 CypA-binding loop) and the entire SIVmac vif gene inserted into the genetic background of HIV-1 NL4-3 (Adachi et al., 1986). From this clone, we have systemically generated a series of HIV-1mt clones as shown in Fig.  5. Because CCR5-tropic (R5) viruses of HIV-1 are thought to be clinically more important than CXCR4-tropic (X4) viruses, we have constructed two sets of HIV-1mt clones. Our strategy for generation of HIV-1mt clones pathogenic for macaques are as follows: (i) Adaptation of viruses in macaque cells. Targets for infection are cynomolgus and rhesus macaque lymphocyte cell lines immortalized by Herpesvirus saimiri (HVS) ( Table 4).; (ii) In vitro mutagenesis of the clones based on bioinformatics. With the aid of the computational sciences, new viral genome sequences are designed.; (iii) Selection of appropriate clones by their replication kinetics in macaque lymphocyte cell lines in Table 4. Viruses which replicates similarly with or more robustly than SIVmac239 in cynomolgus and rhesus peripheral blood mononuclear cells are then chosen. On the basis of this strategy, we have successfully obtained a number of new generations with increasing ability to replicate from the original prototype NL-DT5R (see below). However, so far, none of the HIV-1mt clones tested are pathogenic for macaques (pig-tailed and cynomolgus) Nomaguchi et al., manuscript in preparation;Saito et al., 2011), although they all can replicate in the monkeys. The newest clones in Fig. 5 (Shibata et al., 1991), X4-tropic HIV-1 NL4-3 (Adachi et al., 1986) and R5-tropic HIV-1 NF462 (Kawamura et al., 1994a), respectively. HIV-1mt clones on the left and right are X4 and R5 viruses, respectively. Arrows indicate the site of each single/double nucleotide-mutation introduced (Nomaguchi et al., manuscripts in preparation). There are several single-nucleotide mutations in the green area of Gag-CA (Nomaguchi et al., unpublished). h6/7L, Loop between helices 6 and 7.
In parallel with the generation and characterization of a series of HIV-1 mt clones, we have searched for and established macaque cell lines suitable for our projects. Table 4 lists the cell lines we routinely use now. Since the lymphocyte cell lines immortalized by HVS do not lose their original characteristics as primary lymphocytes in most cases and are readily maintained for experiments, to biologically characterize viruses like HIV-1, it is quite important for laboratory researchers to have HVS-immortalized cell lines. In our laboratory, cynomolgus HSC-F (Akari et al., 1996;Fujita et al., 2003) and rhesus M1.3S (Doi et al., 2011) (Doi et al., 2010;our unpublished results). For the polymorphism of TRIM5 alleles, see the references (Newman et al., 2006;Virgen et al., 2008;Wilson et al., 2008).
We have repeatedly examined the replication kinetics of HIV-1mt clones in various macaque cell lines. Fig. 6 shows the typical kinetics (a schema) based on the results from our numerous infection experiments. In highly sensitive HSC-F cells, all the viruses do replicate to distinct extents. As is clear, MN4Rh-3V and MN5Rh-3V replicate most robustly among HIV-1mt clones. In relatively resistant M1.3S cells, three clones do replicate but the others do not. In both cell lines, SIVmac239 (MA239N) (Doi et al., 2010) displays the best potential to replicate. These results indicate that we still need to improve MN4Rh-3V and MN5Rh-3V to obtain the ideal clone, the pathogenic HIV-1mt. In this situation, there are two directions. These are the selection of host macaques susceptible to the currently available clones and the further efforts to obtain the desired clones. First, pig-tailed and/or the other macaque species sensitive to the viruses can be selected by their TRIM5 alleles (Newman et al., 2006;Virgen et al., 2008;Wilson et al., 2008), and used for infection. Indeed, American research groups have adopted this strategy using the pig-tailed macaques/variants of simian-tropic (st) HIV-1 with a vif-substitution only Thippeshappa et al., 2011). However, we very much prefer to take the second possibility. Through this approach, we would be able to better understand the molecular mechanism underlying various events between the pathogen and host. Furthermore, if one is interested in the studies to analyze the mutations, adaptations, and evolution of the pathogen, the pressure-giving environment (Malim & Emerman, 2008), i.e., natural hosts having a wide variety of restriction factors, would be much better. Of a particular note, pig-tailed monkeys infected with various st HIV-1s have not yet develop AIDS Hatziioannou et al., 2009;Thippeshappa et al., 2011).

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Macaque-Tropic HIV-1 Derivatives: A Novel Experimental Approach to Understand Viral Replication and Evolution In Vivo 339 Fig. 6. Schematic representation of replication kinetics of various viral clones. A schema of replication kinetics is illustrated. Molecular proviral clones for study are shown on the right. Routinely, cell-free virus samples are prepared by transfection of proviral clones into 293T cells (Kamada et al., 2006), and viruses produced in cells of equal RT units are inoculated into HSC-F and M1.3S cells (Table 4). After infection, viral replication is monitored at intervals by RT activity in the culture supernatants.

Conclusion
We have described the generation of CXCR4-tropic and CCR5-tropic HIV-1 clones with macaque cell-tropism (HIV-1mt) in this chapter. The best X4 and R5 viruses we have now replicate comparably with a standard SIVmac clone in macaque cells, although their pathogenicity for macaques needs to be determined. The genomes of these HIV-1 mt clones contain the entire vif gene of SIVmac, some nucleotide substitutions in the gag gene to give a small number of mutated amino acids, two adaptive mutations in the pol gene, and one adaptive mutation in the env gene (Fig. 5).