Distinct requirements for HIV-1 accessory proteins during cell coculture and cell-free infection

Anastasia Zotova, Anastasia Atemasova, Alexey Pichugin, Alexander Filatov, Dmitriy Mazurov

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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.

Original languageEnglish (US)
Article number390
Issue number5
StatePublished - May 2019
Externally publishedYes

Bibliographical note

Funding Information:
Funding: Most of the experiments on design and generation of HIV mutants (Figure 1), infectivity evaluation (Figure 3), BST2 knockout generation (Figure 5), and microscopy examination (Figure 6) were funded by the grant of Russian Science Foundation (RSF 15-15-00135). The experiments on 293T/CD4/X4 cell line generation and HIV-1 replication in these cells (Figure 2) and MOI evaluation (Figure 4) were supported later on by the grant RSF 18-14-00333.

Publisher Copyright:
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.


  • Accessory proteins
  • BST2
  • Cell-to-cell infection
  • CRISPR-Cas9 knockout
  • HIV-1
  • Nef
  • Restriction factors
  • Vpu


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