In the present study, we sought to investigate the biological role of residues between the two domains of HIV-1 CA (Y145-L151) () and in particular, to assess their importance for virus assembly and replication. Our approach was to make point mutations in residues encompassing the entire region, i.e., alanine substitutions in all residues except P147, which was changed to leucine. The results clearly demonstrate that the CA interdomain linker is crucial for facilitating proper core stability and architecture.
Analysis of mutant phenotypes shows that the mutants can be divided into three classes: (i) infectious virions (S146A, T148A); (ii) completely noninfectious virions with severe defects in viral morphology and function (Y145A, I150A, L151A); and (iii) virions with very low infectivity, having an attenuated phenotype (P147L, S149A) (). Interestingly, the infectivity data for some of the mutants can be correlated with what is known about CA structure in this region. Recently, a study combining high resolution NMR analysis of recombinant HIV-1 CA containing residues 144–231 with cryo-EM data for tubular assemblies of this protein revealed the importance of residues Y145, T148, and L151 for CTD dimer formation (Byeon et al., 2009
). Residue Y145 is crucial for formation of this interface and for biological function, since Y145A or Y145F mutants are not infectious and have defects in core stability and assembly (Byeon et al., 2009
), consistent with our Y145A data (; –; and ) (see below). Moreover, of all the mutants studied here, Y145A is the only one that exhibits a CypA deficiency (), presumably as a result of global changes in CA folding. A similar defect was also observed with two severely defective N-terminal CA mutants (W23A and F40A) (Tang et al., 2003b
). A significant reduction in CypA incorporation would be expected to be associated with mutated residues in the NTD, although our data do not rule out such an effect when mutation of a residue near the NTD also results in global disruption of CA structure. The apparent discrepancy concerning the localization of Y145 is likely due to differences in the techniques and protein constructs used for the structure determinations. An additional possibility, although highly speculative, is that CA might adopt different conformations that place Y145 in either the NTD or CTD, depending on the particular stage in the virus life cycle.
Residues L151 and T148 (in order of importance) also exhibit intermolecular interactions that are involved in CTD dimer formation (Byeon et al., 2009
) (I.L. Byeon, personal communication). Thus, the lack of infectivity of L151A is consistent with the structural data (). Most likely, retention of the WT phenotype in the case of T148A () is due to the conservative change from threonine to alanine. The S146A mutation may not impact infectivity because this residue is exposed on the surface of the protein (A.M. Gronenborn, personal communication).
As mentioned above, a major conclusion that emerges from our study is that residues Y145-L151, with the possible exception of S146, are critical for proper core formation. Indeed, point mutations that lead to abrogation or severe reduction of infectivity are often associated with formation of virion cores that are highly unstable: retention of CA in core fractions is negligible (Y145A, I150A, L151A) or only slightly higher than background (P147L, S149A), whereas ~40% of total CA in the sample is associated with WT, S146A, and T148A cores under our conditions (). Core instability of S149A was also observed in studies on HIV-1 CA residues that are potential substrates for phosphorylation (Brun et al., 2008
; Wacharapornin et al., 2007
). Reduction of RT activity associated with unstable mutant cores is less severe than depletion of CA in the case of P147L and S149A (). Thus, removal of the mutant CA shells might occur shortly after entry by premature uncoating (Stremlau et al., 2006
); reviewed in (Arhel, 2010
; Levin et al., 2010
), whereas RT might be less labile due to its location in the interior of the virus. In accord with the CA sedimentation data, we also find that P147L, S149A, and I150A are unable to saturate TRIMCyp in the assay for abrogation of host restriction (), which gives a positive readout only if the cores have optimal stability (Forshey et al., 2005
A striking result of this work is the novel finding that despite the poor replication capacity of P147L and S149A, infectivity can be rescued in an efficient and specific manner by pseudotyping env−
virions with VSV-G ( and ). Similar results were obtained for S149A and S178A in one of the studies of CA phosphorylation (Brun et al., 2008
). Additionally, these authors showed that the pseudotyped virions undergo more efficient viral DNA synthesis. In contrast, VSV-G pseudotyping does not rescue the infectivity of the three noninfectious mutants in our study ().
It is generally thought that HIV-1 entry into cells depends on interaction with cellular receptors followed by fusion at the plasma membrane (Hunter, 1997
). However, there is also evidence supporting the possibility that entry occurs by an endosomal pathway (Miyauchi et al., 2009
). With respect to pseudotyping with VSV-G, it is known that VSV-G-mediated virus entry occurs via pH-dependent endocytosis (Matlin et al., 1982
), but the reason why this mode of entry can result in rescue of infectivity is not known. We speculate that endocytosis might allow delivery of the cores to the nuclear pore without premature uncoating and a requirement to traverse cytoplasmic microfilament/microtubule networks (Arhel et al., 2007
; Bukrinskaya et al., 1998
; McDonald et al., 2002
), thereby bypassing the core instability defect. A somewhat similar suggestion was made in an earlier study on VSV-G pseudotyping of nef
-defective HIV-1 virions (Aiken, 1997
). Alternatively, some other aspect of VSV-G-mediated entry, e.g., faster fusion kinetics with VSV-G than with HIV-1 Env (Hulme et al., 2011
; Iordanskiy et al., 2006
), might result in bypass of the infectivity defect conferred by the P147L and P149A mutations. Collectively, these considerations lead us to suggest that the two mutants might be useful tools for studies on the viral entry pathway.
The P147L and S149A assembly properties and core architecture are also of great interest. Thus, despite the instability of P147L and S149A cores (), a significant number of mutant virions contain conical cores that are indistinguishable from WT structures in images generated by TEM (; ). Moreover, in contrast to the Y145A () (Byeon et al., 2009
), Y145F (Byeon et al., 2009
), and I150A () CA proteins, P147L and S149A CA form tubular assemblies that are very similar to the long tubes assembled by WT CA, although some subtle differences can be detected by TEM (). Experiments exploiting higher resolution EM techniques are being initiated in an effort to identify potential ultrastructural differences between WT and mutant core structures.
It is now established that in addition to other defects, virions having unstable or hyperstable cores are unable to undergo or complete reverse transcription in infected cells (Aiken, 2006
; Bowzard et al., 2001
; Forshey et al., 2002
; Tang et al., 2001
; Tang et al., 2003b
; von Schwedler et al., 2003
). Accordingly, we find that the noninfectious mutant I150A makes ~104
less viral DNA products than WT and no late products (). However, in agreement with the EM analysis, synthesis of viral DNA products by the P147L and S149A mutants is reduced by only 10-fold compared with WT and all of the expected DNA products are detected ().
Taken together, our results raise the following question: “How can we reconcile our finding that in two assays affected by core stability, P147L and S149A give negative results ( and ), whereas in other assays reflecting core structure, assembly, and reverse transcription activity (, , and ; ), these mutants exhibit an attenuated phenotype?” It would appear that differences between the WT and mutant CA protein structures, which in turn dictate core structure and biological activity (reviewed in (Adamson and Freed, 2007
; Vogt, 1997
), are subtle and may not be sufficient to generate a positive readout in some assays. This is most likely the case in the TRIMCyp abrogation assay (). Data from a recent structural study support the idea that the hexagonal scaffold of TRIM5α proteins is assembled using the symmetry and spacing of the CA lattice as a template (“pattern recognition”) (Ganser-Pornillos et al., 2011
). Thus, even subtle defects in CA structure and especially defects that lead to core instability (Forshey et al., 2005
) could prevent significant binding of TRIM5α proteins to CA. Additionally, the low recovery of CA from isolated mutant cores is not entirely unexpected, since sedimentation at high speeds, even for relatively short times, is likely to lead to disruption of cores that are unstable. In contrast, EM and PCR analysis of DNA synthesis involve more gentle treatment of virions, thereby generating a modulated response in these assays.
These findings have important implications for understanding the molecular nature of HIV-1 assembly, since they underscore the unusual plasticity of CA, which despite the rigorous structural requirements that govern assembly and integrity of viral cores, permits some expression of biological activity even under less than optimal circumstances (Tang et al., 2007
). Additional studies on the structure of the P147L and S149A CA proteins should be invaluable for elucidating the ultrastructure of HIV-1 conical cores and its relation to CA function.
In summary, we have shown for the first time that residues between the NTD and CTD of HIV-1 CA (Y145-L151) have a crucial role in virus assembly and formation of conical cores. Mutations in three of these residues (Y145A, I150A, and L151A) lead to a total loss of infectivity, defects in core morphology and stability, as well as virtually complete abrogation of viral DNA synthesis. However, two mutants (P147L and S149A), while poorly infectious, exhibit an attenuated phenotype, including rescue of infectivity by pseudotyping with VSV-G, modest ability to undergo reverse transcription, and assembly of cores with seemingly normal architecture. These findings provide new insights into the biological function of a region in HIV-1 CA that has only recently become the subject of intense interest and represents a potential target for anti-HIV therapy.