Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection

Abstract

The role of accessory proteins during cell-to-cell transmission of HIV-1 has not been explicitly defined. In part, this is related to difficulties in measuring virus replication in cell cocultures with high accuracy, as cells coexist at different stages of infection and separation of effector cells from target cells is complicated. In this study, we used replication-dependent reporter vectors to determine requirements for Vif, Vpu, Vpr, or Nef during one cycle of HIV-1 cell coculture and cell-free infection in lymphoid and nonlymphoid cells. Comparative analysis of HIV-1 replication in two cell systems showed that, irrespective of transmission way, accessory proteins were generally less required for virus replication in 293T/CD4/X4 cells than in Jurkat-to-Raji/CD4 cell cocultures. This is consistent with a well-established fact that lymphoid cells express a broad spectrum of restriction factors, while nonlymphoid cells are rather limited in this regard. Remarkably, Vpu deletion reduced the level of cell-free infection, but enhanced the level of cell coculture infection and increased the fraction of multiply infected cells. Nef deficiency did not influence or moderately reduced HIV-1 infection in nonlymphoid and lymphoid cell cocultures, respectively, but strongly affected cell-free infection. Knockout of BST2—a Vpu antagonizing restriction factor—in Jurkat producer cells abolished the enhanced replication of HIV-1 ΔVpu in cell coculture and prevented the formation of viral clusters on cell surface. Thus, BST2-tethered viral particles mediated cell coculture infection more efficiently and at a higher level of multiplicity than diffusely distributed virions. In conclusion, our results demonstrate that the mode of transmission may determine the degree of accessory protein requirements during HIV-1 infection.

Environmental Restrictions: A New Concept Governing HIV-1 Spread Emerging from Integrated Experimental-Computational Analysis of Tissue-Like 3D Cultures

“…Since the restriction of virus spread by extracellular tissue architecture has only recently begun to become apparent, we can only speculate about the effector function mediating the restriction. Such environmental restrictions could include direct effects on the pathogen, such as the impairment of cell-free infectivity [92] (see below), but could also impact the host cells (e.g., via the promotion of longer-lasting cell contacts with architecture optimized for virus transmission). It can also be envisioned that, for example, transcriptional and epigenetic changes resulting from mechanosensing of the cells in the 3D environment would affect expression and activity of pro-and antiviral host cell factors.…”

Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41

“…Supernatants containing PVs were harvested and cleared through 0.45-μm-pore-size filters at 54 h posttransfection. The 293T/CD4 or Raji/CD4 cells that we described previously ( 67 ) were first infected with PVs to express CCR5. After immunostaining with respective MAb and positive sorting, cells were transduced to express one of the gp41 peptides.…”

Engineering T cell resistance to HIV-1 infection via knock-in of peptides from the heptad repeat 2 domain of gp41

“…Supernatants containing PVs were harvested and cleared through 0.45 μm pore size filters at 54 h posttransfection. The 293T/CD4 or Raji/CD4 cells that we described previously (68) were, first, infected with PVs to express CCR5. After immunostaining with respective mAb and positive sorting, cells were transduced to express one of gp41 peptides.…”

“…HIV-1 cell-free and cell coculture infections. For HIV-1 single-round cell-coculture and cell-free infection tests with intron-regulated reporter vector inLuc, please, refer to our previous papers (39,40,68). Briefly, to generated viral particles pseudotyped with different Envs, 293T cells in 10-cm dish were cotransfected with 4 μg of pCMVΔ8.2 R plasmid, 6 μg of pUCHR-inLuc-mR reporter vector, and 1 μg of one of the HIV-1 Env expressing vectors pIIINL4env (a gift from Dr. Eric Freed, NCI-Frederick, USA), pJRFLenv (69) and pZM135 (a gift from Drs.…”