Mannose receptor is a restriction factor of HIV in macrophages and is counteracted by the accessory protein Vpr

HIV-1 Vpr is necessary to support HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is the restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a macrophage mannose-MR response system to facilitate infection while evading MR’s normal role, which is to trap and destroy mannose-expressing pathogens.


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Vpr is a highly conserved HIV accessory protein that is necessary for optimal replication in 63 macrophages (Balliet, Kolson et al. 1994) but its mechanism of action is poorly understood.

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Studies using human lymphoid tissue (HLT), which are rich in both T cells and macrophages,

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Identification of restriction factor counteracted by Vpr in primary human monocyte-derived 123 macrophages. Because we had previously determined that Vpr functions in macrophages to 124 counteract a macrophage selective restriction factor that targets Env, we reasoned that Env-125 binding proteins selectively expressed in macrophages were potential candidate restriction 126 factors. To determine wether any factors fitting this description were targeted by Vpr, we 127 performed western blot analysis of candidate Env binding proteins in MDM matched for wild type 128 or Vpr null HIV infection frequency ( Figures 1A and B). We found that one such protein, mannose 129 receptor (MR), which is highly expressed on macrophages and has been previously shown to

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To determine whether the modest effect of Nef alone was due to using HIV to deliver Nef as 166 compared to an adenoviral vector delivery system used in a prior publication (Vigerust, Egan et

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While the effect of Nef has been previously reported and found to be due to disruption of MR 176 intracellular trafficking (Vigerust, Egan et al. 2005  that Vpr in HEK293T cells had no effect on expression of MR controlled by a heterologous CMV 189 promoter ( Figure 2G and S1). Thus, we concluded that Vpr does not degrade MR by the direct 190 mechanism it uses to degrade UNG2 and SMUG1.

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In addition to targeting proteins for degradation, Vpr also functions to inhibit transcription of genes

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Combined effect of Vpr and Nef dramatically enhances Env levels in primary human MDM.

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To determine whether the striking downmodulation of MR we observed with expression of both 206 Nef and Vpr affected viral spread in MR + macrophages, we generated additional mutations in HIV-207 1 89.6 to create a nef-null mutant and a vpr-nef-null double mutant. As expected, in transfected 208 HEK293T cells these mutations did not alter Env protein levels ( Figure 3A Figure 4C, a fragment of the YU-2 genome containing most of env but none of vpr 250 ( Figure 4C, shaded portion) was cloned into NL4-3 and NL4-3 vpr-null. As expected, these genetic 251 alterations did not affect Env protein levels or virion release in transfected HEK293T cells ( Figure   252 4D and E). To confirm that the chimeric Env was still functional, we examined infectivity in T cells 253 prior to performing our analyses in primary human MDM. Conveniently, sequence variation within 254 the gp120 region allows YU-2 Env to only utilize the co-receptor CCR5 for entry, whereas NL4-3 255 can only utilize CXCR4. Thus, we expected the NL4 3env YU2 chimera would switch from being

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To determine whether swapping a limited portion of YU-2 containing Env into NL4-3 alleviated 265 the requirement for Vpr, we examined Env expression and virion release in primary human MDM 266 infected with these viruses. Because the parental NL4-3 virus required pseudotyping with a 267 macrophage-tropic Env for entry and was unable to spread in MDM, all infections were treated 268 with entry inhibitors AMD3100 and maraviroc 48 hours after inoculation to block subsequent 269 rounds of infection. Remarkably, we observed that wild type NL4-3 Env but not chimeric NL4-3 270 env YU2 required Vpr for maximal expression ( Figure 4G). Moreover, MDM infected with the 271 chimeric HIV had a reduced requirement for Vpr for maximal virion release ( Figure 4H). This

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To confirm that mutation of N230 and N339 disrupted the mannose patch on Env, we assayed 291 the ability of 2G12, which recognizes epitopes in the mannose patch (Sanders, Venturi et al. 2002, 292 Scanlan, Pantophlet et al. 2002), to neutralize wild type and mutant Env. As shown in Figure 5D, 293 wild type but not mannose deficient N230D N339E was neutralized by 2G12. In addition, we found 294 that these substitutions did not disrupt infection of a T cell line that does not express MR ( Figure   295 5E). However, somewhat unexpectedly, we found that HIV containing the N230D N339E Env 296 substitutions were approximately 40% less infectious to primary human macrophages expressing 297 MR than the wild type parental virus ( Figure 5E). This macrophage-specific difference in infectivity 298 suggested that mannose on Env facilitates initial infection through interactions with MR, which is 299 highly expressed on differentiated macrophages. To examine this possibility further, we asked 300 whether mannan, which competitively inhibits MR interactions with mannose containing glycans 301 (Shibata, Metzger et al. 1997), was inhibitory to HIV infection. As a negative control, we tested 89.6 ∆env pseudotyped with vesicular stomatitls virus G-protein Env (VSV-G) which has only two 303 N-linked glycosylation sites, both of which contain complex-type rather than high-mannose 304 glycans (Reading, Penhoet et al. 1978) and therefore should not bind MR or be inhibited by 305 mannan. As expected, we found that infection of a T cell line lacking MR was not sensitive to 306 mannan ( Figure 5F, left panel). However, infection of MDM by wild type HIV-1 was inhibited up to 307 16-fold by mannan. This was specific to HIV Env because mannan did not inhibit infection by HIV 308 lacking env and pseudotyped with the heterologuos VSV-G Env ( Figure 5F). Interestingly, 309 mannan also inhibited baseline macrophage infection by mannose-deficient Env (89.6 Env N230D 310 N339E), indicating that N230D N339E substitutions did not completely abrogate glycans on Env 311 that are beneifical to initial infection. In sum, our results demonstrate that interactions with 312 mannose binding receptors are advantageous for initial HIV infection of macrophages and that 313 the glycans remaining on Env N230D N339E retain some ability to bind glycan receptors on 314 macrophages that facilitate infection.

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While interactions between high-mannose residues on Env and MR are advantageous for viral 317 entry, we hypothesized that they interfered with intracellular Env trafficking and were deleterious 318 to egress of Env-containing virions in the absence of Vpr and/or Nef. To test this, we examined 319 virion release and Env expression by HIVs encoding the mannose-deficient Env N230D N339E 320 plus or minus Vpr expression. We found that mannose-deficient Env had a reduced requirement 321 for Vpr for maximal virus relase compared with the parental wild type virus in a spreading infection 322 system ( Figure 5G, p<0.001). In addition, the mannose-deficient Env had a reduced requirement 323 for both Nef and Vpr in virion release assays using primary human MDM infected for a single

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Finally, we asked whether the mannose-deficient Env had increased stability in primary human 331 MDM lacking Vpr and/or Nef by western blot analysis. Remarkably, we found that the mannose-

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Remarkably, we observed that silencing MR markedly reduced Env restriction -once differences 343 in infection frequency as assessed by Gag pr55 expression were accounted for ( Figure 6A).

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These results support the conclusion that the Env restriction observed in vpr-null 89.6 is 345 dependent on expression of MR.  whether MR was responsible for these defects in MDM lacking Vpr, we measured Vpr-dependent 352 HIV-1 spread from primary human MDM silenced for MR to activated primary T cells freshly 353 isolated from the same donor. In this assay system, co-cultured cells were stained for CD3 to 354 distinguish T cells and CD14 to distinguish MDM as shown in Figure S2. Indeed, we found that 355 silencing MR dramatically reduced the requirement for Vpr to support spread from MDM to T cells 356 ( Figure 6D). In addition, MR silencing reduced the need for Vpr in virus release assays in the co-357 culture system ( Figure 6E). These data support the conclusion that MR is the previously identified  inoculated briefly (6 hours) and cultured for two days, REJO produced detectable infection but 371 CH077 did not ( Figure 7A, lower panel). Using the experimental protocol diagrammed in Figure   372 7B, we observed that when T cells were cultured for two days with cell free virus without 373 spinnoculation, both T/F viruses failed to infect a significant fraction of T cells ( Figure 7C, upper 374 panel). In contrast, when T cells were co-cultured for two days with MDM that had previously been 375 infected as in Figure 6F, the T/F virus REJO was transmitted to T cells but CH077 was not (

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Monocytes were plated at 5x10 5 cells/well in a 24 well dish, except for those to be transduced 514 with lentivirus and puromycin selected, which were plated at 1 x10 6 cells/well.