Macrophages archive HIV-1 virions for dissemination in trans

Abstract

Viruses have evolved various strategies in order to persist within the host. To date, most information on mechanisms of HIV-1 persistence has been derived from studies with lymphocytes, but there is little information regarding mechanisms that govern HIV-1 persistence in macrophages. It has previously been demonstrated that virus assembly in macrophages occurs in cytoplasmic vesicles, which exhibit the characteristics of multivesicular bodies or late endosomes. The infectious stability of virions that assemble intracellularly in macrophages has not been evaluated. We demonstrate that virions assembling intracellularly in primary macrophages retain infectivity for extended intervals. Infectious virus was recovered directly from cytoplasmic lysates of macrophages and could be transmitted from macrophages to peripheral blood lymphocytes in trans 6 weeks after ongoing viral replication was blocked. Cell-associated virus decayed significantly from 1 to 2 weeks post infection, but decreased minimally thereafter. The persistence of intracellular virions did not require the viral accessory proteins Vpu or Nef. The stable sequestration of infectious virions within cytoplasmic compartments of macrophages may represent an additional mechanism for viral persistence in HIV-1-infected individuals.

Figure 1

Cytoplasmic assembly of HIV-1 is maintained in macrophages in the absence of a spreading infection. (A) Experimental scheme. Monocyte-derived macrophages were infected with VSV-G-pseudotyped, X4-tropic HIV-1 variants containing an intact (HIV-1_WT) or a defective envelope gene (HIV-1_ΔEnv). At weekly intervals, cell-associated and extracellular gag p24 levels were determined (B) and at 2, 4 and 6 weeks post infection, the presence of morphogenically mature HIV-1 particles in cytoplasmic vesicles was determined by electron microscopy (C).

Figure 2

Stability of cell-associated virus in the absence of de novo viral gene expression. (A) Experimental design. HIV-1_2Δteto contains a bacterial tet promoter, and proviral gene expression is dependent upon the presence of DOX. Macrophages were infected with VSV-G-pseudotyped HIV-1_2Δteto in the presence of DOX. After 4 days, de novo viral gene expression was terminated by removal of DOX. (B) Levels of cell-associated gag p24 in macrophages maintained in the presence of DOX (DOX 0–44) or in macrophages in which DOX was removed 4 days after infection (DOX 0–4).

Figure 3

Potent suppression of spreading HIV-1 replication in macrophages by IVS. Macrophages were infected with an R5-tropic HIV-1 variant (HIV-1_ADA) and maintained in IVS at the indicated concentrations. Viral replication was monitored by measurement of RT activity in culture supernatants.

Figure 4

Intracellular virions capable of initiating T-cell infection in trans persist in macrophages. (A) Macrophages were infected with a VSV-G-pseudotyped X4-tropic HIV-1 variant (HIV-1_LAI). At 16 h and 7 days post infection, further de novo production of infectious virus was blocked by IVS. At the indicated intervals, culture supernatants were examined for the presence of mature virions containing processed gag p24 by ELISA (B). At 4 and 7 weeks post infection, the presence of infectious virus, able to initiate lymphocyte infection in trans, was determined after addition of activated PBLs. Infection in trans (C) was determined by PCR quantitation of 2-LTR circle cDNAs in PBLs. Data shown are representative of four independent experiments.

Figure 5

Decay characteristics of cell-associated virus in long-term macrophage cultures. (A) Macrophages were infected with VSV-G-pseudotyped HIV-1_LAI and 4 days after infection, de novo virus production was blocked by addition of IVS. At the indicated time intervals, the presence of processed gag p24 in culture supernatants (B, left panel) was determined and the presence of infectious cell-associated virus was evaluated in trans following addition of activated PBLs (B, right panel). Data shown are representative of three independent experiments.

Figure 6

Role of the accessory protein Nef in the maintenance of cell-associated virus in long-term macrophage cultures. (A) Experimental scheme. Macrophages were infected with VSV-G-pseudotyped HIV-1 variants harboring intact (HIV-1_WT) or mutated (HIV-1_Δnef) nef genes. At 4 days after infection, cells were maintained in the presence or absence of IVS. At the indicated intervals, extracellular gag p24 was determined by ELISA (B), and extracellular infectious virions were titered on MAGI cells (C). The presence of infectious cell-associated virus was determined following addition of PBLs and measurement of infectivity in trans at 3, 5 and 7 weeks post-infection (D). Data shown are representative of three independent experiments.

Figure 7

The accessory protein Vpu is required for an efficient spreading infection in macrophages but not for maintenance and viral dissemination in trans of cell-associated virus. (A) Requirement for Vpu in a spreading viral infection of macrophages. Macrophages were infected with R5-tropic HIV-1_ADA variants comprising intact (ADA_WT) or mutated (ADA_ΔVpu) vpu genes. Viral replication in macrophages was assessed by measurement of RT activity in culture supernatants. (B) Role of Vpu in the maintenance of cell-associated virus was examined using the same strategy as in Figure 6. Macrophages were infected with VSV-pseudotyped X4-tropic HIV-1 variants containing functional or inactive vpu genes. IVS was then added 4 days post infection to inhibit virus production. At 4 and 6 weeks post infection, the presence of processed gag p24 in culture supernatants and in cell lysates was determined by ELISA (C). (D) Infectivity of cell-associated virus at 4 and 6 weeks post infection was determined following addition of PBLs and measurement of infectivity in trans. 2-LTR cDNAs were quantitated in two aliquots (0.1 and 0.5) of infected PBL lysates. (E) The presence of infectious virus in cytoplasmic macrophage lysates was directly assessed by titration on MAGI cells. 2-LTR cDNA products were quantitated in two aliquots (0.1 and 0.5) of MAGI cell extracts. Data shown are representative of two independent experiments.