SIV infection is endemic in wild-living apes throughout equatorial Africa, yet only certain lineages of SIVcpz and SIVgor have been found to infect and cause disease in humans (1
). This has raised the question of whether certain strains of SIVcpz/SIVgor are more prone than others to cross from apes to humans. Although all SIVcpz and SIVgor strains identified to date grow efficiently in human CD4+
T cells (6
), these maximally activated cultures are unlikely to represent a reliable surrogate of zoonotic potential. In the present study, we show that 4 genetically diverse SIVcpz strains from both central and eastern chimpanzees are capable of establishing a productive infection in HLT, the major site of HIV-1 replication in vivo. This is remarkable, given the number of different host proteins and innate restriction factors that primate lentiviruses must utilize or counteract (11
) and suggests that most (if not all) SIVcpz strains have the biological properties required for cross-species infection of humans. However, replication fitness of SIVcpz doubled upon the introduction of a previously identified human signature at position 30 of the viral matrix protein. Thus, even chimpanzee-to-human transmission seems to impose substantive adaptive hurdles to ape lentiviruses.
Although replacement of Met/Leu with Arg at Gag30 enhanced the replication potential of all 4 SIVcpz strains in HLTs, their overall growth rates still remained 3-fold lower than those of the HIV-1 strains, indicating additional adaptations. One of these likely includes effective antitetherin activity. Tetherin (also termed Bst-2) inhibits the release of virions from infected cells by “tethering” them at the plasma membrane (35
). Like most primate lentiviruses, SIVcpz uses its Nef protein to antagonize tetherin (37
). However, the SIVcpz Nef protein is inactive against human tetherin due to a 5–amino acid deletion in the cytoplasmic domain of the human protein that confers resistance to Nef binding (37
). This lack of antitetherin activity must explain, at least in part, why SIVcpz constructs containing the human Gag30 adaptation still replicate less efficiently than HIV-1 strains in HLT cultures. In fact, previous studies have shown that a defective vpu
gene decreased replication of HIV-1 in ex vivo–infected human tonsillary tissue by about 3-fold, although it was not determined whether this reduction was due to lack of antitetherin activity or other Vpu functions, such as degradation of CD4 (40
). Nonetheless, the results suggest that effective tetherin antagonism may be required for efficient replication of SIVcpz in HLTs.
Current data suggest that a fitness gain at Gag30 was a prerequisite for the emergence of HIV-1 groups M, N, and O. The fact that only 1 of the 2 recently discovered HIV-1 group P strains has switched from a Met to a Lys at Gag30 is interesting, since this could indicate that this group is still in the process of adaptation (41
). However, a single amino acid change at Gag30 seems easier to achieve than the acquisition of a new antitetherin function. Pandemic HIV-1 group M was able to master this by switching from Nef- to Vpu-mediated tetherin antagonism (38
). However, this did not happen in HIV-1 groups O and P, whose Vpu proteins failed to gain this function (38
). The Vpu protein of HIV-1 group N gained some antitetherin activity, but lost its ability to degrade CD4. Thus, it is tempting to speculate that the adaptive change in Gag30 was a necessary first step to increase the replication potential of SIVcpz in HLTs such that additional mutations could subsequently accumulate to facilitate the gain of antitetherin function by its vpu
Although SIVcpz is endemic in both central and eastern chimpanzees, only SIVcpzPtt
strains have thus far been found in humans (1
). It is possible that humans are less frequently exposed to SIVcpz-infected eastern chimpanzees or that SIVcpzPts
strains have been transmitted but gone unrecognized. However, it is also possible that SIVcpzPts
strains are inherently less fit to infect humans. We found that the SIVcpzPts
strain TAN2 produced about 2-fold lower viral titers in HLTs than the SIVcpzPtt
strains MT145 and MB897 (Figure ). Moreover, changing Gag30 from Lys to Leu, which is found in TAN2, impaired the replication of HIV-1 NL4-3 in HLT more severely than a change to Met found in all SIVcpzPtt
strains (Figure ). However, not all SIVcpzPtt
strains replicated efficiently in HLT (Figure ), and a recent molecular epidemiological study in the Democratic Republic of the Congo revealed that a number of SIVcpzPts
strains encode Met at Gag30, as found in SIVcpzPtt
(B.H. Hahn, unpublished observations). Thus, neither poor replication potential in HLTs nor coding differences at Gag30 can explain the absence of SIVcpzPts
zoonotic infections. Further studies will need to define whether certain adaptations, such as the acquisition of antitetherin activity, may be more difficult to achieve for viruses endemic in eastern chimpanzees.
The mechanism or mechanisms of the Gag30-mediated effect on viral replication fitness remain to be determined. The MA protein is not only a structural component of the virion, but is also involved in viral entry/uncoating, cytoskeletal-mediated transport, nuclear import, virion assembly, and incorporation of the viral envelope glycoprotein into progeny virions (44
), interacting with as many as 20 different cellular proteins (34
). These include 3 of the 4 clathrin adaptor protein complexes, AP-1, AP-2, and AP-3 (46
), the microtubule-associated cellular motor protein KIF4 (49
), calmodulin (50
), 47-kDa tail interacting protein (TIP47) (51
), elongation factor 1-α (EF1α) (52
), the virion-associated nuclear shuttling protein (VAN) (53
), and the barrier-to-autointegration factor (BAF) (54
). Among these proteins, calmodulin, EF1α, BAF, AP-1 μ1A chain, and AP-2 μ2 chain are all predicted to be identical between chimpanzees and humans and thus unlikely to be responsible for the species-specific selection pressure. The chimpanzee and human AP-3 δ chain, TIP47, and VAN proteins differ by 3 or 4 amino acids, respectively, while the KIF4 motor protein is the most divergent, exhibiting 7.2% amino acid difference between human and chimpanzee homologs. Previous studies have shown that the AP-3 δ chain is involved in Gag trafficking, TIP47 mediates Env incorporation into particles, and VAN and KIF4 may play a role during postentry steps (48
). It is possible that Gag30, which is located in one of the N-terminal helices of MA, interacts with one or more of these proteins or with as-yet-unidentified host factors and may thus influence steps early in the viral life cycle. It should be noted, however, that the magnitude of the effect of Gag30 on viral fitness is strain dependent. Thus, as-yet-unidentified changes in the MA protein or elsewhere in the viral genome might minimize or even remove the need for a basic residue at Gag30.
The fact that we found a much less pronounced effect of Gag30 on the growth potential of SIVcpz in human CD4+
T cell cultures is not surprising. Fully activated CD4+
T cells represent optimal, albeit artificial, targets for viral replication. For example, they may express MA-interacting proteins at levels different from target cells that are not fully activated by exogenous stimuli. T cell activation is known to be associated with complex changes of the plasma membrane, the cytoplasm, the cytoskeleton and at the nucleus (55
), all of which may minimize the dependency on MA protein functions. The mode of virus spread in the culture, i.e., cell-free versus cell-cell, may also have an impact on the magnitude of the Gag30 function. Perhaps most importantly, effective spread of HIV-1 and SIVcpz in vivo involves a complex interplay between different types of virally infected and noninfected cells, which are more accurately represented by lymphoid tissues than stimulated CD4+
T lymphocyte cultures. Thus, our finding that a basic residue significantly increases the replication fitness of SIVcpz strains in HLT strongly suggests that it is also relevant for viral replication and transmission in vivo.
In summary, our results demonstrate that nonadapted SIVcpz strains can replicate to some extent in HLTs. We also show that the acquisition of a basic residue at Gag30 in the viral matrix protein significantly increased the replication fitness of SIVcpz in the new human host, albeit not to the levels of current HIV-1 group M strains. Thus, additional changes, such as the development of effective tetherin antagonism, were required to achieve full replication fitness. Since exposures of humans to ape lentiviruses are likely to continue, further analyses of host-specific adaptation will be important to assess the future human zoonotic risk.