Complex determinants within the Moloney murine leukemia virus capsid modulate susceptibility of the virus to Fv1 and Ref1-mediated restriction

Abstract

Two of the most well-known genetic mechanisms in mammalian cells which control the susceptibility of cells to productive infection by retroviruses and lentiviruses rely on the cellular Fv1 and Ref1 restriction factors, which act, after viral entry, to prevent productive infection through their interactions with viral capsid (CA) sequences. While previous studies of Fv1 restriction involving N- and B-tropic murine leukemia viruses (MLVs) had demonstrated that the identity of a single amino acid residue at CA110 (arginine vs. glutamic acid) determines whether the resulting virus is N (arg) or B-tropic (glu), analogous studies of dual-tropic MLVs, such as Moloney MLV (Mo-MLV), have shown that additional residues other than CA110 are also involved in the specification of dual-tropic host range. Here we have further studied the CA determinants of Mo-MLV host range, with an emphasis on identifying additional CA residues and unique combinations of CA residues which differentially influence the ability of the resulting virus to infect murine and human cells. First, we show that CA82, a residue previously identified to affect the pattern of Fv1 restriction of different MLV viruses in murine cells, is a particularly strong potentiator of B-tropism in an Mo-MLV background carrying a glutamic acid residue at CA110 (A110E substitution), and that interestingly, different residues at CA82 lead to distinct patterns of restriction in human but not in murine cells. We also identify another CA residue, CA214, as a similarly potent potentiator of B-tropism, in the context of the A110E substitution. While another substitution at CA110, A110R, leads to strong potentiation of N-tropism in murine cells, in the absence of additional mutations, we found that A110R alone was not sufficient to confer appreciable restriction in Ref1-expressing cells, despite the fact that authentic N-MLV shows strong restriction in those cells. In conjunction with the A110R substitution, substitutions at CA82, but not at CA214, do lead to significant restriction in human cells, thus demonstrating a distinction between the interactions between those two determinants of restriction and CA110. Finally, using cell lines engineered to express the TRIM5alpha(hu) gene product, recently identified as the Ref1 restriction factor, and RNAi technology to knock-down expression of TRIM5alpha(hu) in human cells, we directly demonstrate that the unique patterns of restriction observed in human cells with the different mutants are consistent with a TRIM5alpha(hu)-mediated restriction. These studies shed further light on the complex determinants within the viral CA gene product which control the susceptibility of murine and human cells to retroviral infection.

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

“…4A and Fig. S4), including the β1/β2 hairpin, L5/6, and L4/5 regions in HIV CA and the corresponding sites on MLV CA (30,33,(45)(46)(47)(48)(49)(50). Specifically, TRIM5α V1 may interact with CA β1/β2 at the center of the CA hexamer, as swapping the V1 region of rhTRIM5α and huTRIM5α resulted in a change of the restriction specificity toward MLV CA with mutations in the β1/β2 region (30).…”

The Requirement for Nucleoporin NUP153 during Human Immunodeficiency Virus Type 1 Infection Is Determined by the Viral Capsid

“…Infection assays utilized single-round viruses carrying either green fluorescent protein (GFP) or luciferase reporter genes. GFP-based constructs included equine infectious anemia virus (EIAV) (56,57), HIV-1 (31), Rous sarcoma virus (RSV) (12) (Addgene plasmid 13878; obtained from Constance Cepko via Addgene, Cambridge, MA), FIV (36,46), MLV (58,68), Mason-Pfizer monkey virus (MPMV) (55), HIV-2 strain ROD (HIV-2 ROD ), and simian immunodeficiency viruses from Macaca mulatta (SIV MAC ), Chlorocebus sabaeus (SIV AGM Sab), and Chlorocebus tantalus (SIV AGM Tan) (82). Luciferasebased viruses included MLV/HIV-1 LAI chimeras (75,76) and HIV-1 NL4-3 -derived D64N/D116N (N/N) IN active site and Vpr and/or DNA flap mutants of pNLX.Luc (45,47).…”

Novel Postentry Resistance to AKV Ecotropic Mouse Gammaretroviruses in the African Pygmy Mouse, Mus minutoides

“…The three resistance alleles of Fv1 target specific residues in the virus capsid. Capsid amino acid 110 distinguishes N-and B-tropic viruses (25), but various substitutions in amino acid positions 82 to 117 of the CA N-terminal domain can generate NR-or NB-tropic viruses (12,20,27,39,44).…”

“…CA110 is the target for Fv1 n (E110) and Fv1 b (R110) restriction (25), and MLVs bearing R110, A110, and H117 are restricted by human TRIM5␣ (49). Studies on naturally occurring or mutated MLVs with altered tropism have identified at least eight additional positions within the CA82-214 segment that can alter sensitivity to Fv1 or TRIM5␣, and these tropism-determining residues can be masked or modulated when combined (12,20,27,39,44). A similar ability to target multiple capsid residues or combinations of residues may also explain why the 2-amino-acid Minr target identified in AKV MLV (R110 L117) was not present in three of the six restricted viruses.…”

“…A single amino acid at CA position 110 distinguishes the N-and B-tropic viruses restricted by Fv1 n and Fv1 b (25). Additional mutagenesis studies identified targets for Fv1 NR and NB tropism at additional sites in the N-terminal domain of CA (12,20,27,39,44). Despite the identification of the Fv1 gene and its target, the mechanism of virus restriction has not been determined.…”