In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail - PubMed (original) (raw)

In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail

P N Fultz et al. J Virol. 2001 Jan.

Abstract

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxphi) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (DeltaGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239DeltaGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239DeltaGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxphi signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.

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Figures

FIG. 1

FIG. 1

Partial amino acid sequences of the membrane-spanning and cytoplasmic domains of SIVmac239 and Tyr-721 mutants. Tyr-721 is indicated by the arrow. Residues that are critical to the formation of the endocytosis signal include Gly-720, Tyr-721, and Val-724 (8, 9).

FIG. 2

FIG. 2

Growth curves of SIVmac239 and Tyr-721 mutants. Stocks of SIVmac239, SIVmac239Y/I, and SIVmac239ΔGY were normalized for RT activity and used to infect CEMx174 cells (A) or phytohemagglutinin-stimulated PBMC from rhesus macaques (B). Viral replication was quantified by serial determination of RT activity in culture supernatants. The experiments were performed twice for CEMx174 and three times for PBMC.

FIG. 3

FIG. 3

Changes in viral burdens and hematologic parameters in macaques inoculated with SIVmac239 and Tyr-721 mutants. Animals designated by a dagger in the plasma RNA panels either died or were euthanized due to SIV-induced immunodeficiency. Plasma RNA levels were determined by the bDNA assay, which had levels of sensitivity of either 104 or 1,500 copies/ml, depending on the time at which the assay was performed. Some samples were tested by QC RT-PCR, for which the sensitivity was 300 copies/ml. All points below the dotted lines were negative by both assays, with the exception of two values marked by an asterisk. These values were 350 and 800 copies/ml, respectively, for AGP at 4 weeks and N8G at 20 weeks after infection.

FIG. 4

FIG. 4

Humoral immune responses of macaques infected with SIVmac239 and Tyr-721 mutants. (A to C) Serum antibody titers after infection of macaques with SIVmac239 (A), SIVmac239Y/I (B), or SIVmac239ΔGY (C) are shown. The titers are expressed as the reciprocal of the highest dilution of serum yielding a value above the cutoff for the ELISA. (D) Neutralizing-antibody titers against H9-adapted SIVmac251 and SIVmac239 grown in rhesus PBMC are shown for serum samples collected 16 weeks after infection.

FIG. 5

FIG. 5

Molecular evolution of SIVs recovered from animals infected with the SIVmac239 and Tyr-721 mutants. Peripheral blood DNA from rhesus macaques infected with SIVmac239 (A), SIVmac239Y/I (B), or SIVmac239ΔGY (C) was extracted, amplified by PCR, cloned, and sequenced. Partial amino acid sequences of Env in cells from individual animals infected with each virus are shown. For panels B and C, the sequence of the mutant virus used for inoculation is shown relative to SIVmac239. Wk, weeks following inoculation; C, code designation for individual clones. Amino acid identity to SIVmac239 is indicated by a dash, deletions are indicated by a dot, and a stop codon (found only in week 14, clone 4, from AGP) is shown by an asterisk. In each panel, the YRPV sorting signal is underlined.

FIG. 5

FIG. 5

Molecular evolution of SIVs recovered from animals infected with the SIVmac239 and Tyr-721 mutants. Peripheral blood DNA from rhesus macaques infected with SIVmac239 (A), SIVmac239Y/I (B), or SIVmac239ΔGY (C) was extracted, amplified by PCR, cloned, and sequenced. Partial amino acid sequences of Env in cells from individual animals infected with each virus are shown. For panels B and C, the sequence of the mutant virus used for inoculation is shown relative to SIVmac239. Wk, weeks following inoculation; C, code designation for individual clones. Amino acid identity to SIVmac239 is indicated by a dash, deletions are indicated by a dot, and a stop codon (found only in week 14, clone 4, from AGP) is shown by an asterisk. In each panel, the YRPV sorting signal is underlined.

FIG. 6

FIG. 6

Comparative evaluation of virus replication in competition assays in CEMx174 cells. (A) PCR analysis of SIVmac239 versus SIVmac239ΔGY competition. The env region was PCR amplified from cell lysates harvested on the indicated days. The larger band (158 bp) corresponds to the wild-type virus, and the smaller band (152 bp) corresponds to the mutant virus. (B) FRET analysis of SIVmac239 versus SIVmac239ΔGY competition. The samples from panel A were subjected to melting-curve analysis. Plasmid controls 1 and 2 correspond to SIVmac239 and SIVmac239ΔGY, respectively. The early, middle, and late time points are days 1, 11, and 35, respectively. (C) FRET analysis of SIVmac239ΔGY versus SIV-7-14 competition. Plasmid controls 1 and 2 correspond to SIVmac239ΔGY and SIV-7-14, respectively. The early, middle, and late time points are days 4, 7, and 17, respectively. (D) FRET analysis of SIVmac239 versus SIVmac239Y/I competition. Plasmid control 1 and 2 correspond to SIVmac239 and SIVmac239Y/I, respectively. The early, middle, and late time points are days 7, 17, and 30, respectively.

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