摘要

SERINC蛋白，是丝氨酸插入蛋白的简称，在鞘磷脂和磷脂酰丝氨酸等以丝氨酸为原料的脂质合成中发挥着生理功能。SERINC家族蛋白在真核生物中高度保守，其中酵母编码1种SERINC蛋白，人编码5种SERINC蛋白。SERINC家族蛋白结构上高度保守，都由9到11个跨膜区构成。在5种SERINC蛋白中，SERINC3和SERINC5发现具有抑制HIV-1感染的活性，其中SERINC5的抑制HIV-1感染的活性更强。目前关于SERINC5抑制HIV-1的机制尚无定论，一种解释是SERINC5和HIV-1的膜结构相互作用从而抑制HIV-1的感染性；另一种解释是SERINC5位于细胞膜上，在病毒颗粒释放的过程中，掺入到病毒膜上从而抑制病毒再次感染时病毒颗粒与细胞膜融合的过程。除此之外，对于不同的病毒膜蛋白，例如VSVG和Ebola GP，SERINC5并没有抑制活性，并且SERINC5对HIV-1的不同亚型抑制活性也不相同，这些问题仍然得不到合理的解释。因此我们借助另外一种包膜病毒甲型流感病毒，在研究SERINC5是否抑制流感感染的同时，探求SERINC5抑制病毒的作用机制以及差异原因。

甲型流感病毒目前仍然是全球健康的主要威胁。甲型流感病毒包括8段单股负链基因组RNA，可以编码多达11个蛋白。流感病毒需要吸附到细胞表面启动感染过程，在流感病毒HA与宿主细胞膜表面的唾液酸受体结合以后，病毒颗粒通过内吞进入细胞内部。一旦进入细胞内体，低pH激活M2离子通道，引起HA构象改变，驱动病毒与内体脂膜的融合，病毒的基质蛋白M1游离到胞内。病毒包膜与细胞膜融合释放流感病毒核糖核酸蛋白vRNP进入细胞质并运输到细胞核内进行转录、翻译、复制。

在我们的研究过程中发现，SERINC5确实存在抑制流感病毒感染的活性。首先SERINC5可以抑制HA/NA假病毒颗粒的感染性。在产毒细胞中，过表达SERINC5不影响HIV-1假病毒颗粒的产生，上清通过超速离心收集病毒颗粒可以检测到SERINC5的掺入。将相同p24量的病毒上清感染HIV-1的报告细胞系TZM-BL检测荧光信号，发现SERINC5可以抑制WSN膜结构HA/NA的感染性，提示SERINC5可以抑制流感病毒的感染。接着采用A549过表达SERINC5以及敲除SERINC5的细胞系，感染正常的流感病毒WSN株，发现SERINC5可以抑制WSN单轮以及多轮感染。

进一步对SERINC5抑制流感病毒WSN的作用机制的研究，发现SERINC5通过抑制流感病毒感染的早期膜融合过程，从而抑制病毒感染。对于不同流感HA亚型的假病毒，我们发现SERINC5能够抑制低糖基化型的流感病毒HA的感染。因此我们认为SERINC5对不同膜蛋白的抑制活性的差异在于膜蛋白糖基化水平的高低。

综上所述，我们全面深入的研究了SERINC5在流感病毒感染过程中的具体作用，首次证明了SERINC5可以通过抑制病毒与细胞膜融合的过程从而抑制流感病毒的感染，以及SERINC5对不同膜蛋白的抑制活性的差异在于糖基化水平的高低，为SERINC5的抗病毒机制提供了更加深入的证据，同时为包膜病毒的致病机理，以及抗病毒机制的研究提供了重要参考。

SERINC5 inhibits influenza virus membrane fusion

Abstract

SERINC proteins are short for serine incorporator plays a role in the synthesis of serine-containing lipids such as sphingomyelin and phosphatidylserine. SERINC are highly conserved in eukaryotes from yeast (encode one gene) to humans (encode five genes). SERINC homologues share a highly similar topology, they are integral membrane proteins with 9 to 11 membrane spanning regions. Among five SERINC family members SERINC3 and SERINC5 were identified as inhibitors of HIV-1 infectivity of which SERINC5 has a stronger inhibitory activity. Possible mechanisms of fusion inhibition by SERINC5 are an open question. One model is that SERINC5 interacts with Env trimers and inhibits their activity which is supported by the observation that SERINC5 increases the susceptibility of HIV-1 Env to certain antibodies. The other is that virion-incorporated SERINC5 might delay fusion of the virion and target cell membranes. In addition, there is still no reasonable explanation why HIV-1 with VSVG, Ebola GP and some resistant env glycoproteins, but not with sensitive retrovirus pathogens, makes HIV-1 resistant to SERINC5. Therefore, we sought another enveloped virus influenza A virus to investigate whether SERINC5 inhibits influenza infection, as well as the mechanism by which SERINC5 inhibits virus infection and the reasons for the difference.

Influenza A virus (IAV) infection is still a major threat to global health. Influenza A virus contains 8 segments of negative single-stranded genomic RNA which encodes for up to 11 proteins. Influenza A virus initiates the infection process with influenza attachment. Influenza hemagglutinin (HA) binds to the target cell via sialic acid linkages on host glycoproteins. After HA-mediated binding to the receptor, virion endocytosis is triggered. Once within endosomes, the low pH activates the M2 ion channel and causes conformational change in HA that drive fusion of the viral and endosomal lipid membranes, viral matrix protein M1 dissociates from the inner membrane to the viral envelope. Fusion of the two membranes release influenza viral ribonucleoproteins (vRNPs) into the cytoplasm and transport to the nucleus for transcription, translation and replication.

Here we demonstrate the idea that SERINC5 restricts influenza A virus infection. First, SERINC5 inhibits the infectivity of WSN pseudoparticles with HA/NA. In virus producing cells, the overexpression of SERINC5 did not affect the production of HIV-1 pseudoparticles and the supernatant can detect the incorporation of SERINC5 by ultracentrifugation. Luciferase value was detected in HIV-1 reporter cell lines TZM-BL infected with HIV-1 pseudoparticles, we found that SERINC5 could inhibit the infectivity of HIV-1 pseudoparticles with HA/NA from influenza WSN, suggesting that SERINC5 could inhibits the infection of influenza virus. Moreover, using SERINC5 overexpress or knockout A549 cell lines, we find that SERINC5 can inhibit influenza A virus WSN infection with single cycle or multicycle infection.

Further research shows that SERINC5 inhibit influenza infection during early steps of virus entry, which precise influenza virus membrane fusion. Also, we find that SERINC5 can inhibit low N-glycosylation subtypes of influenza HA infectivity indicating that the difference in the inhibitory activity of SERINC5 to different membrane proteins lies in the level of N-glycosylation.

From what has been discussed above, we studied the specific role of SERINC5 in the process of influenza virus infection. We proved that SERINC5 can inhibit influenza virus and cell membrane fusion process so as to inhibit influenza virus infection for the first time. We also proved that the differences of inhibitory activity of SERINC5 targeting different membrane protein lies on the levels of N-glycosylation. Which provides further evidence for the research of SERINC5 antiviral mechanisms. And also provides an important reference for the research on mechanism of envelope virus pathogenic and antiviral response.