Determinants Flanking the CD4 Binding Loop Modulate Macrophage Tropism of Human Immunodeficiency Virus Type 1 R5 Envelopes

Program in Molecular Medicine and Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Journal of Virology (Impact Factor: 4.44). 02/2009; 83(6):2575-83. DOI: 10.1128/JVI.02133-08
Source: PubMed

ABSTRACT Human immunodeficiency virus type 1 R5 viruses vary extensively in phenotype. Thus, R5 envelopes (env) in the brain tissue of individuals with neurological complications are frequently highly macrophage-tropic. Macrophage tropism correlates with the capacity of the envelope to exploit low CD4 levels for infection. In addition, the presence of an asparagine at residue 283 within the CD4 binding site has been associated with brain-derived envelopes, increased env-CD4 affinity, and enhanced macrophage tropism. Here, we identify additional envelope determinants of R5 macrophage tropism. We compared highly macrophage-tropic (B33) and non-macrophage-tropic (LN40) envelopes from brain and lymph node specimens of one individual. We first examined the role of residue 283 in macrophage tropism. Introduction of N283 into LN40 (T283N) conferred efficient macrophage infectivity. In contrast, substitution of N283 for the more conserved threonine in B33 had little effect on macrophage infection. Thus, B33 carried determinants for macrophage tropism that were independent of N283. We prepared chimeric B33/LN40 envelopes and used site-directed mutagenesis to identify additional determinants. The determinants of macrophage tropism that were identified included residues on the CD4 binding loop flanks that were proximal to CD4 contact residues and residues in the V3 loop. The same residues affected sensitivity to CD4-immunoglobulin G inhibition, consistent with an altered env-CD4 affinity. We predict that these determinants alter exposure of CD4 contact residues. Moreover, the CD4 binding loop flanks are variable and may contribute to a general mechanism for protecting proximal CD4 contact residues from neutralizing antibodies. Our results have relevance for env-based vaccines that will need to expose critical CD4 contact residues to the immune system.

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Available from: Dennis R Burton, Sep 26, 2015
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    • "This dogma has been challenged by work demonstrating that R5 tropic envelopes are diverse in their ability to replicate in monocyte derived macrophages [8]. More specifically, envelope glycoproteins (Envs) from viruses isolated from central nervous system often confer efficient macrophage replication upon pseudotyping viruses as compared to Envs from blood or lymph nodes in patients with neurocognitive disease [9,10], and since then a diverse range of envelope determinants have been implicated in this phenotype, often associated with the CD4 binding site [11-13]. "
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    ABSTRACT: It is now known that clinically derived viruses are most commonly R5 tropic with very low infectivity in macrophages. As these viruses utilize CD4 inefficiently, defective entry has been assumed to be the dominant restriction. The implication is that macrophages are not an important reservoir for the majority of circulating viruses. Macrophage infection by clinical transmitted/founder isolates was 10-100 and 30-450 fold less efficient as compared to YU-2 and BaL respectively. Vpx complementation augmented macrophage infection by non-macrophage tropic viruses to the level of infectivity observed for YU-2 in the absence of Vpx. Augmentation was evident even when Vpx was provided 24 hours post-infection. The entry defect was measured as 2.5-5 fold, with a further 3.5-10 fold block at strong stop and subsequent stages of reverse transcription as compared to YU-2. The overall block to infection was critically dependent on the mechanism of entry as demonstrated by rescue of infection after pseudotyping with VSV-G envelope. Reverse transcription in macrophages could not be enhanced using a panel of cytokines or lipopolysaccharide (LPS). Although the predominant block to clinical transmitted/founder viruses is post-entry, infectivity is determined by Env-CD4 interactions and can be rescued with VSV-G pseudotyping. This suggests a functional link between the optimal entry pathway taken by macrophage tropic viruses and downstream events required for reverse transcription. Consistent with a predominantly post-entry block, replication of R5 using viruses can be greatly enhanced by Vpx. We conclude therefore that entry is not the limiting step and that macrophages represent clinically relevant reservoirs for 'non-macrophage tropic' viruses.
    Retrovirology 03/2014; 11(1):25. DOI:10.1186/1742-4690-11-25 · 4.19 Impact Factor
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    • "It appears that our CD4-independent variants exist constitutively in or spontaneously acquire the CD4-bound conformation, even in the absence of CD4 [9]. Therefore, loss of coreceptor use plasticity may be specific to Envs that have a pre-formed or spontaneously exposed coreceptor binding site [5-8] rather than Envs that scavenge low levels of CD4 on the cell surface [3,4,31-34]. Taken together, these data suggest that diverse mechanisms exist by which virus can expand its host range into CD4-low or negative cells, which may have different consequences for coreceptor interactions. "
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    ABSTRACT: HIV and SIV generally require CD4 binding prior to coreceptor engagement, but Env can acquire the ability to use CCR5 independently of CD4 under various circumstances. The ability to use CCR5 coupled with low-to-absent CD4 levels is associated with enhanced macrophage infection and increased neutralization sensitivity, but the additional features of these Envs that may affect cell targeting is not known. Here we report that CD4-independent SIV variants that emerged in vivo in a CD4+ T cell-depleted rhesus macaque model display markedly decreased plasticity of co-receptor use. While CD4-dependent Envs can use low levels of macaque CCR5 for efficient entry, CD4-independent variants required high levels of CCR5 even in the presence of CD4. CD4-independent Envs were also more sensitive to the CCR5 antagonist Maraviroc. CD4-dependent variants mediated efficient entry using human CCR5, whereas CD4-independent variants had impaired use of human CCR5. Similarly, CD4-independent Envs used the alternative coreceptors GPR15 and CXCR6 less efficiently than CD4-dependent variants. Env amino acids D470N and E84K that confer the CD4-independent phenotype also regulated entry through low CCR5 levels and GPR15, indicating a common structural basis. Treatment of CD4-dependent Envs with soluble CD4 enhanced entry through CCR5 but reduced entry through GPR15, suggesting that induction of CD4-induced conformational changes by non-cell surface-associated CD4 impairs use of this alternative co-receptor. CD4 independence is associated with more restricted coreceptor interactions. While the ability to enter target cells through CCR5 independently of CD4 may enable infection of CD4 low-to-negative cells such as macrophages, this phenotype may conversely reduce the potential range of targets such as cells expressing low levels of CCR5, conformational variants of CCR5, or possibly even alternative coreceptors.
    Retrovirology 11/2013; 10(1):133. DOI:10.1186/1742-4690-10-133 · 4.19 Impact Factor
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    • "However, unlike CD4 usage, no alteration in CCR5 usage of LT5 Envs was found suggesting that coreceptor binding sites exposure on gp120 post CD4 binding was similar but it substantially varied when gp120 was not engaged by CD4, evident from 17b MAb binding to Env trimers expressed on 293T cells. Our study also highlighted presence of a rare substitution in the V3 loop (I315F) in the LT5.J4b Env (but absent in other LT5 Envs) which was previously shown to modulate susceptibility of HIV-1 Envs to anti-V3 MAbs [42], [44]. Interestingly, the determinants in Env found to modulate the sensitivity of HIV-1 to IgG1b12 or sCD4 did not show any impact on susceptibility to the VRC01 MAb; a broad and a very potent CD4bs neutralizing MAb [16]. "
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    ABSTRACT: Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones). The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.
    PLoS ONE 10/2012; 7(10):e46713. DOI:10.1371/journal.pone.0046713 · 3.23 Impact Factor
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