Studies On The Anti-viral Mechanism Of Feline Host Restriction Factors BST-2 And BCA2

Human BST-2 exhibits broad anti-viral activity against a wide range of enveloped viruses,such as HIV-1,HIV-2,SIV and other retroviruses,as well as Lassa,Marburg and Ebola virus-like particles.During the late phase of the viral replication pathway,BST-2 causes nascent viruses to remain trapped at the surface of the infected cell and to accumulate thereafter in endosomes following internalization.Some reports have claimed several distinct functions of the cytoplasmic tail domain of human BST-2.The deletion of each structural domain on the N-terminal HA-tagged human BST-2 was initially reported to result in severe impairment of its anti-HIV-1 virus release function.However,recent studies indicated that f BST-2 has a very short N-terminal cytoplasmic region compared with those of other mammalian and non-mammalian homologs.However,f BST-2 contains a 57 in-frame nucleotide sequence upstream of the first ATG,starting with a GAG codon that can potentially encode 19 amino acids(f BST-2* peptide),which displayed significant homology with the N-terminal regions of all the other BST-2s analyzed.Surprisingly,it was shown to significantly block budding of FIV,HIV-1,HIV-2 ROD10,SIVmac239 and RD-114 particles.An artificial f BST-2* mutant containing a longer N-terminal cytoplasmic homolog only exhibited limited anti-viral capability.Moreover,ovine BST-2A,one of the two ovine BST-2 isoforms,also retains a cytoplasmic tail shorter than that of human BST-2.Interestingly,this shorter isoform was found to display a stronger anti-viral activity than the longer isoform.Cocka and Bates showed that naturally occurring variants of human BST-2 initiating from the internal ATG were partially resistant to Vpu.Another recent study showed that the cytoplasmic tail of human BST-2 is unnecessary for restricting HIV-1 but important for inducing intracellular signaling,and an N-terminal or internal HA-tagged BST-2 was found to cause the loss of functions of anti-virus release and NF-?B activation.Based on these observations,we hypothesized that the shorter N-terminal cytoplasmic region of the f BST-2 protein may lack the ability to induce NF-?B activation,and the HA tag can also compromise the functions of f BST-2.In this study,our results provide evidence that although the f BST-2 has a much shorter N-terminal cytoplasmic region compared with those of other mammalian and non-mammalian homologs,it still retained anti-viral activity against HIV-1 and FIV pseudovirus viral particle release.However,the lost amino acid sequence was demonstrated to have the ability to mediate activation of NF-?B.Furthermore,fusion of a peptide tag to different positions on the f BST-2 protein could impair its anti-viral activity to varying degrees.The HA tag and f BST-2* peptide in the N-terminal cytoplasmic peptide may have affected the association of the recombinant BST-2 with lipid membranes such as ER retention.Such impairment in intracellular trafficking reduced localization of the N-terminally tagged f BST-2-NHA to the cell surface,eventually weakening its anti-viral function.Meanwhile,mutating both N-glycosylation sites of f BST-2(f BST-2 N79/ 119A)greatly reduced its anti-viral activity,but the influence of the single site mutant(f BST-2 N79 A and f BST-2 N119A)was slight.Importantly,these observations demonstrate that the f BST-2* peptide was abandoned through the process of evolution,as it had a minimal impact on wild-type f BST-2 and even maintained an ability to activate NF-?B.While the anti-viral function,N-glycosylation and ability to activate NF-?B of f BST-2 may be three independent biological functions,its anti-viral activity is related to its cell surface expression.These results provide additional details and implications for the structure-function model of BST-2.Additionally,these findings highlight the importance of the choice of tag position in functional studies of biological molecules.Breast cancer associated gene 2(BCA2),also known as ring finger protein 115(RNF115)or Rabring7,belongs to the E3 ubiquitin ligase family with a ring finger domain,which is overexpressed in more than 50% of infiltrating breast cancers compared with normal tissues and can be associated with proliferation of breast cancer cells in vitro.Overexpression of BCA2 increases the proliferation of NIH3T3 fibroblasts,while si RNA inhibits the growth of BCA2-expressing breast cancer cells.The BCA2 protein has autoubiquitination activity,which depends on its RING domain and the mutation in K26 and K32 lysine in the BCA2 zinc finger(BZF)domain also eliminate autoubiquitination activity.Meanwhile,BCA2 E3 ligase has been shown to be an important factor in regulating the migration of breast cancer cells.Although it has been widely accepted that E3 enzyme can play an active role in the carcinogenesis process,it is less well understood how deregulation of an E3 may occur.BCA2 is a RING type E3 ubiquitin ligase,which was identified as a co-factor that promotes the lysosomal degradation of HIV-1 virions trapped by BST-2,and also acts as a BST-2 independent anti-HIV factor by targeting Gag for lysosomal degradation and restricting HIV-1 transcription by inhibiting the NF-?B pathway.However,the anti-viral mechanism of feline BCA2(f BCA2)is less clear.In this study,the f BCA2 was identified from feline cells,which belongs to the conserved E3 ubiquitin ligase family.The RING domain at C-terminal of f BCA2 is essential for its autoubiquitination.Overall,these results indicated that f BCA2 has similarity of autoubiquitination character with human BCA2,may play an important role in feline mammary carcinoma,which will help to study breast cancer in the cat models.Next,we investigated the anti-viral effect of f BCA2 against HIV-1 as well as the anti-viral function of h BCA2 and f BCA2 against FIV-?Vif-?Orf A-?Env-GFP pseudovirus.We found that there are some similarities and differences between the f BCA2 and h BCA2 anti-HIV-1 mechanisms.The similarities are: both of the two proteins are capable of degrading the HIV-1 Gag protein and are also capable of restricting the transcription of HIV-1 by inhibiting the activation of NF-?B by HIV-1.The difference is that h BCA2 can induce lysosomal pathway degradation of HIV-1 virus particles,whereas f BCA2 does not have this function.In addition,when we investigated the mechanism of inhibition of BCA2 against FIV-?Vif-?Orf A-?Env-GFP pseudovirus,we found that the mechanism of BCA2 protein inhibition of HIV-1 is somewhat different: BCA2 can not induce lysosomal pathway degradation of FIV-?Vif-?Orf A-?Env-GFP pseudovirus particles;BCA2 protein also failed to mediate the degradation of FIV Gag protein.However,BCA2 protein is also able to restrict the transcription of FIV pseudovirus by inhibiting the activation of NF-?B by FIV.These findings suggest that the main mechanism of the BCA2 protein anti-viral function may be to inhibit viral transcription,while the degradation of Gag protein and the inducing of viral particle degradation of lysosomal pathways are both auxiliary functions.In addition,the ring finger domain of the BCA2 protein is essential for its ability to degrade HIV-1 Gag protein,but does not result in the complete loss of function that inhibits viral transcription.These results will contribute to the understanding of the BCA2 anti-viral mechanism,thereby contributing to the establishment of an animal model for the study of HIV infection.Since FIV and HIV-1 have similarities in genomic structure,transmission mechanisms,infection processes,pathogenicity,and antagonism of host factors,and domestic cats also have host-like restriction factors that interfere with FIV infection.Therefore,the domestic cat is considered to be a study of human acquired immunodeficiency syndrome(AIDS)and the construction of treatment strategies related to natural animal models,the cat immunodeficiency virus and cat host restriction factor interaction between the study will be the establishment of cat HIV-1 animal model provides theoretical support,with theoretical and practical significance.In conclusion,the study of the interaction between feline immunodeficiency virus and feline host restriction factor will provide theoretical support for the establishment of feline HIV-1 animal model,which has theoretical and practical significance.