Simian immunodeficiency virus utilizes human and sooty mangabey but not rhesus macaque STRL33 for efficient entry - PubMed (original) (raw)

Simian immunodeficiency virus utilizes human and sooty mangabey but not rhesus macaque STRL33 for efficient entry

S Pöhlmann et al. J Virol. 2000 Jun.

Abstract

It has been established that many simian immunodeficiency virus (SIV) isolates utilize the orphan receptors GPR15 and STRL33 about as efficiently as the chemokine receptor CCR5 for entry into target cells. Most studies were performed, however, with coreceptors of human origin. We found that SIV from captive rhesus macaques (SIVmac) can utilize both human and simian CCR5 and GPR15 with comparable efficiencies. Strikingly, however, only human STRL33 (huSTRL33), not rhesus macaque STRL33 (rhSTRL33), functioned efficiently as an entry cofactor for a variety of isolates of SIVmac and SIV from sooty mangabeys. A single amino acid substitution of S30R in huSTRL33 impaired coreceptor activity, and the reverse change in rhSTRL33 greatly increased coreceptor activity. In comparison, species-specific sequence variations in N-terminal tyrosines in STRL33 had only moderate effects on SIV entry. These results show that a serine residue located just outside of the cellular membrane in the N terminus of STRL33 is critical for SIV coreceptor function. Interestingly, STRL33 derived from sooty mangabeys, a natural host of SIV, also contained a serine at the corresponding position and was used efficiently as an entry cofactor. These results suggest that STRL33 is not a relevant coreceptor in the SIV/macaque model but may play a role in SIV replication and transmission in naturally infected sooty mangabeys.

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Figures

FIG. 1

FIG. 1

SIVmac239 does not use rhSTRL33 for efficient entry. 293T cells were cotransfected with plasmids expressing human or macaque CD4 and the indicated entry cofactors of human and rhesus macaque origin. At 1 day posttransfection the cells were detached from the plates, seeded in 48-well dishes, and infected in triplicate with intact luciferase reporter viruses containing 50 ng of p27 antigen. Luciferase activities in the cellular extracts were measured at 3 days postinfection. Error bars give standard deviations from average values measured in three independent infections. Similar results were obtained in three additional experiments. As controls the transfected cells were infected with envelope-deleted SIVmac239 (Δenv) or with a chimeric SIV containing the murine leukemia virus glycoprotein (MuSIV) (63). Luciferase values obtained with rhSTRL33 (1,885 ± 61) were about 13-fold lower than those obtained with huSTRL33 (24,043 ± 2,993).

FIG. 2

FIG. 2

Different SIV entry efficiencies are not due to inefficient expression of rhSTRL33. (A) 293T cells were cotransfected with CD4 and expression constructs for wild-type and AU1-tagged huSTRL33 and rhSTRL33. SIVmac239 entry was determined as described in the legend to Fig. 1. The results represent average values of three independent infections. (B) 293T cells were transfected with vectors expressing AU1-tagged huSTRL33 or rhSTRL33. Protein expression was verified by Western blot analysis as described in Materials and Methods. Control, cell extracts derived from mock-transfected 293T cells.

FIG. 3

FIG. 3

Schematic presentation of the N-terminal region and the extracellular domains of rhSTRL33. Amino acid variations in huSTRL33 compared to the rhSTRL33 sequence are indicated. huSTRL33 and rhSTRL33 are 94% identical at the amino acid level, with most differences clustered at the amino terminus (20, 46). Differences between huSTRL33 and rhSTRL33 are indicated by the shaded residues. The numbers give the amino acid positions in rhSTRL33. Bar, cell membrane.

FIG. 4

FIG. 4

Species-specific sequence variations in the N terminus of STRL33 determine SIV coreceptor function. (A) Schematic presentation of the AU1-tagged STRL33 recombinants with an huSTRL33 N terminus and a rhSTRL33 C terminus (huN/rhC) and with an rhSTRL33 N terminus and an huSTRL33 C terminus (rhN/huC). (B) 293T cells were cotransfected with CD4 and expression constructs for the indicated STRL33 variants. SIVmac239 entry was tested as described in the legend to Fig. 1. The results represent average values of three independent infections. Control, cell extracts derived from mock-transfected 293T cells.

FIG. 5

FIG. 5

Amino acid substitution of S31R impairs functional activity of rhSTRL33 as an SIVmac239 entry cofactor. Mutational analysis of rhSTRL33 and huSTRL33. The specific mutations compared to the original rhSTRL33 and huSTRL33 amino acid sequences are shown at the left. Dashes indicate amino acid identity, and dots indicate gaps. The relative entry efficiencies compared to human STRL33 are shown at the right. Average values from three infections are shown, and comparable results were obtained with independent virus stocks.

FIG. 6

FIG. 6

Serine 31 is critical for STRL33 coreceptor function of several SIV isolates. SIVmacBK28, SIVsmΔB670 cl.3, and SIVmac17E/F Env-pseudotyped GFP reporter viruses were used to infect cells coexpressing CD4 and the indicated STRL33 variants. Infection efficiency (percentage of GFP-positive cells) is shown relative to that obtained for huSTRL33 (100%). Comparable results were obtained in two independent experiments.

FIG. 7

FIG. 7

smSTRL33 mediates efficient SIV entry. (A) Sequence variations at the N terminus of STRL33 derived from humans (hu), rhesus macaques (rh), sooty mangabeys (sm), pig tail macaques (ptm), and African green monkeys (agm) (20, 46). Dashes, identity with the human-derived STRL33 sequence; dots, gaps introduced to optimize the alignment. The position of the serine residue, which is critical for STRL33 coreceptor function, is boxed. (B) Entry of luciferase reporter viruses pseudotyped with SIVmac239, SIVmac316, and SIVsmΔB670 cl.3 Env into cells coexpressing CD4 and smSTRL33. Infections were performed as indicated in Materials and Methods. Entry efficiency is shown relative to that obtained for huSTRL33. Data represent average values obtained for three independent infections. Control, cell extracts derived from mock-transfected 293T cells infected with pseudotyped luciferase reporter viruses.

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