CD4-independent entry and replication of simian immunodeficiency virus in primary rhesus macaque astrocytes are regulated by the transmembrane protein

Abstract

Previous studies have demonstrated that the genetic determinants of simian immunodeficiency virus (SIV) neurovirulence map to the env and nef genes. Recent studies from our laboratory demonstrated that SIV replication in primary rhesus macaque astrocyte cultures is dependent upon the nef gene. Here, we demonstrate that macrophage tropism is not sufficient for replication in astrocytes and that specific amino acids in the transmembrane (TM) portion of Env are also important for optimal SIV replication in astrocytes. Specifically, a Gly at amino acid position 751 and truncation of the cytoplasmic tail of TM are required for efficient replication in these cells. Studies using soluble CD4 demonstrated that these changes within the TM protein regulate CD4-independent, CCR5-dependent entry of virus into astrocytes. In addition, we observed that two distinct CD4-independent, neuroinvasive strains of SIV/DeltaB670 also replicated efficiently in astrocytes, further supporting the role of CD4 independence as an important determinant of SIV infection of astrocytes in vitro and in vivo.

FIG. 1.

Replication kinetics of neurovirulent (SIV/17E-Fr) and nonneurovirulent (SIVmac239 and SIV/17E-Cl) SIV and SIV/2-1 (containing the TM of SIV/17E-Fr and the SU and Nef of SIVmac239) in primary rhesus macaque lymphocytes (A), macrophages (B), and astrocytes (C). Data are from one of several independent experiments yielding similar results. Mock, mock-infected control.

FIG. 2.

Diagram of the genetic structure of a panel of SIV recombinant molecular clones used to examine the roles of specific TM sequences for replication in primary macaque lymphocytes, macrophages, and astrocytes. TM proteins diagrammed in black have an Arg residue at position 751, while those shown in gray have Gly at position 751. White bars indicate the presence of a stop codon in the TM CTD or in Nef (SIV/17E-Cl only). LTR, long terminal repeat.

FIG. 3.

Replication kinetics of SIV molecular clones containing mutations in TM in rhesus macaque primary lymphocytes (A), primary rhesus macaque macrophages (B), and primary rhesus macaque astrocytes (C). Data are from one of several independent experiments yielding similar results. Mock, mock-infected control.

FIG. 4.

Effect of soluble CD4 (sCD4) on replication of SIV infectious molecular clones in primary macaque cells. (A) SIV/17E-Fr, SIV/3-11, SIV/3-11RG, SIV/3-7, and SIV/3-7stop virus stocks were preincubated with 1 μg of sCD4/ml for 1 h at 37°C before 50 ng of virus was used to infect cells. The supernatants were collected every 5 days and assayed for viral p27 protein. (B) Induction of virus replication by sCD4 at day 20 postinoculation for the experiment shown in panel A. Fold induction is the log₁₀ p27 value obtained with the sCD4/p27 value obtained without sCD4. (C) sCD4 facilitates replication of SIVmac239 in primary rhesus macaque astrocytes cultures. SIVmac239 was preincubated with sCD4, and the experiment was performed as described for panel A. (D) sCD4 does not enhance SIV/17E-Fr infection of primary rhesus macaque macrophages. Virus stocks were preincubated with 1 μg of sCD4/ml, and 50 ng of virus was used to infect primary macrophage cultures. The supernatants were collected every 2 days and analyzed for the presence of SIV p27 protein. For panels A, B, and C, data are from one of several independent experiments. Mock, mock-infected control.

FIG. 5.

Replication kinetics of SIV/DeltaB670 in primary astrocytes. Cells were incubated with SIV/DeltaB670, washed three times, and incubated at 37°C. Supernatants were collected every 5 days and assayed for the presence of SIV p27 protein. Data are from one of several independent experiments yielding similar results. Mock, mock-infected control.