Productive Replication of vif -Chimeric HIV-1 in Feline Cells

Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCECells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression. T he nuclear pore complex (NPC) represents a key barrier to the replication of many viruses that infect eukaryotic cells (1-3). For retroviruses such as the primate lentivirus human immunodeficiency virus type 1 (HIV-1), the late, productive phases of the viral life cycle require efficient nuclear export of unspliced, and thus intron-containing, viral genomic RNAs (gRNAs) that (i) encode the group-specific antigen (Gag) and Gag-Pol polyproteins that form ...

Section: Species-specific Regulation Of Hiv-1 Rev Activitysupporting

confidence: 80%

Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

Aligeti

Behrens

Pocock

et al. 2014

“…It is believed that FIV Vif makes a fundamental contribution to overcoming the restrictions imposed by fA3Z3 and fA3Z2-Z3 (23,47,63). FIV Vif colocalizes with the feline A3 proteins, targets them for proteasome degradation, and rescues the infectivity of the virus (47). The specific interactions between FIV Vif and the fA3 proteins are still unclear, and the precise mechanism of FIV Vif induced degradation of fA3s remains to be elucidated.…”

mentioning

confidence: 99%

“…Infectivity of Bet-deficient FFV is reduced not only by genomic deamination but also by an A3-induced reduction of particle release (28). The infectivity of Vif-deficient feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) is reduced by fA3Z3 and fA3Z2-Z3 (28,37) by induction of G3A hypermutation of the viral cDNA (37,47). The antiretroviral activities of the fA3Z2s are inhibited by the foamy virus accessory Bet protein (28), while the mechanism of FeLV against fA3s is unknown (38).…”

mentioning

confidence: 99%

“…The antiretroviral activities of the fA3Z2s are inhibited by the foamy virus accessory Bet protein (28), while the mechanism of FeLV against fA3s is unknown (38). It is believed that FIV Vif makes a fundamental contribution to overcoming the restrictions imposed by fA3Z3 and fA3Z2-Z3 (23,47,63). FIV Vif colocalizes with the feline A3 proteins, targets them for proteasome degradation, and rescues the infectivity of the virus (47).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Identification of a Cullin5-ElonginB-ElonginC E3 Complex in Degradation of Feline Immunodeficiency Virus Vif-Mediated Feline APOBEC3 Proteins

Wang¹,

Zhang²,

Lv³

et al. 2011

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominantnegative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

“…These results indicate that binding detected by co-IPs is not a proper reporter for Vif-mediated counteraction affecting the protein levels. Indeed, several previous studies reported that binding of Vif and A3 is not the only determinant for complete Vif-medi- ated degradation (53,57,(83)(84)(85). Kitamura et al identified several residues between ␣2 and ␣3 helices of A3C binding to HIV-1 Vif (63).…”

Section: Discussionmentioning

confidence: 99%

Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C

Zhang

Vasudevan

et al. 2016

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F. IMPORTANCEThe APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein. S imian immunodeficiency virus (SIV) naturally infects manyOld World primate species in Africa. The pandemic of human immunodeficiency virus (HIV) originated from cross-species transmission events of SIVs to humans. HIV-1 was introduced into the human population by multiple transmissions of a chimpanzee (cpz) virus, which is known as SIVcpz. The less virulent human lentivirus, HIV-2, was derived from SIVsmm, which was obtained from sooty mangabey monkeys (sm...