Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses

RSV和HMPV是副粘病毒家族的细胞质被膜RNA病毒。它们的基因组是15.2kb(RSV)或13.3kb(HMPV)的单链负义RNA，编码10个mRNAs和11个独特蛋白质(RSV)或8个mRNAs和9个独特蛋白质(HMPV)。每个病毒编码一个核蛋白N、磷蛋白P、基质蛋白M、小分子疏水蛋白SH、主要糖蛋白G、融合糖蛋白F、聚合酶因子M2-1和M2-2以及聚合酶蛋白L。此外，RSV还编码两种非结构蛋白NS1和NS2。 我们评估了密码子对去最佳化(CPD)策略作为开发遗传和表型稳定的RSV减毒株的一种手段。众所周知，密码子对的使用在自然界中是有偏见的。具体地说，由于遗传密码的简并性，多肽链中的任何给定氨基酸对都有可能由各种不同的同义密码子组合编码，但观察到的密码子对的使用通常偏向于支持可能组合的子集。这种偏见的一个因素被认为是翻译效率和准确性，因为由于tRNA几何结构和其他因素，某些tRNA对的组合在核糖体的A和P位点上是受欢迎的。CPD涉及在蛋白质编码序列的众多位置故意引入表达不足的同义密码子对，以实现次优表达。这些替换只涉及ORF，因此非蛋白质编码的基因组区域不受影响。此外，CPD只涉及同义密码子替换，因此氨基酸编码不受影响。此外，应用于一个或几个基因的CPD通常涉及成百上千个核苷酸的变化，因此对去衰减应该是高度难解的。最近，CPD被应用于脊髓灰质炎病毒和流感病毒，并被证明能产生减毒株。 我们设计了四组CPD RSV基因组，其中指示的ORF被重新编码：(I)Min A；NS1、NS2、N、P、M和SH(即基因组左侧的三分之一)；(Ii)Min B；G和F(位于基因组中部)；(Iii)Min L；L(位于基因组右端)；和(Iv)Min FLC；除M2-1和M2-2外的所有ORF。商业合成重编的基因组区域，构建4株CPD病毒，并通过反向遗传学回收。所有CPD病毒均对温度敏感(敏感度：Min FLC&GT；Min L&GT；Min B&Gt；Min A)。我们推测，CDP可能会减慢翻译速度，从而产生蛋白质折叠问题，而温度升高会加剧蛋白质折叠问题。所有CPD突变体的体外生长效率都低于野生型(Wt)RSV，即使在32℃的允许温度下(生长效率：wt&gt；闽L&gt；闽A&gt；闽FLC&gt；闽B)。因此，G和F ORF的CPD效果最好。CPD病毒在小鼠和非洲绿猴(AGM)中表现出一定的限制性，并诱导对wt RSV的免疫。这项研究确定了RSV的新候选疫苗，并表明非片段负链RNA病毒的CPD可以快速产生具有一系列减毒表型的候选疫苗。 我们使用基因缺失的HMPV毒株来评估附着G和疏水的小分子SH糖蛋白在体外对原代人单核细胞来源的树突状细胞(MDDC)摄取HMPV的作用，以及对MDDC成熟和自体T细胞激活的影响。缺失G和SH(DelSHG)可增强MDDC的感染力，但对MDDC的成熟影响不大。然而，经SHG刺激的MDDC可促进自体Th1极化的CD4+T细胞的增殖。这种效应与病毒复制无关。T细胞增殖的增强严格依赖于病毒刺激的MDDC和CD4+T细胞之间的接触。共聚焦显微镜显示SH和G的缺失与记忆的CD4+T细胞和病毒刺激的MDDC之间的免疫突触数量的增加有关。MDDC对HMPV的摄取主要通过巨噬细胞吞噬。与SHG相比，野生型(WT)病毒的摄取减少，表明SH和G糖蛋白具有抑制作用。此外，DC-SIGN介导的内吞作用提供了一种依赖于SH和/或G的次要替代途径，因此仅对WT起作用。总之，SH和G糖蛋白降低了HMPV被MDDC内化的能力，导致HMPV刺激的MDDC激活CD4+T细胞的能力降低。这项研究描述了一种以前未知的病毒免疫逃避机制。这一点很有趣，因为HMPV的再次感染在一生中很常见，不需要显著的抗原变化，这表明对HMPV的保护性免疫反应是不完整的和短暂的。本研究提供了一种可能有助于抑制宿主免疫的机制。

RSV and HMPV are cytoplasmic enveloped RNA viruses of the paramyxovirus family. Their genomes are single strands of negative-sense RNA of 15.2 kb (RSV) or 13.3 kb (HMPV) that encode 10 mRNAs and 11 unique proteins (RSV) or 8 mRNAs and 9 unique proteins (HMPV). Each virus encodes a nucleoprotein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, major glycoprotein G, fusion glycoprotein F, polymerase factors M2-1 and M2-2, and the polymerase protein L. In addition, RSV encodes two nonstructural proteins NS1 and NS2. We evaluated the strategy of codon-pair deoptimization (CPD) as a means of developing genetically and phenotypically stable attenuated RSV strains. It is well known that there is a bias in codon-pair usage in nature. Specifically, any given pair of amino acids in a polypeptide chain has the possibility to be encoded by a variety of different combinations of synonymous codons due to the degeneracy of the genetic code, but the observed usage of codon-pairs typically is biased to favor a subset of the possible combinations. One factor in this bias is thought to be translational efficiency and accuracy, because certain combinations of tRNA pairs are favored at the A and P sites in the ribosome due to tRNA geometry and other factors. CPD involves the deliberate introduction of under-represented synonymous codon-pairs into numerous sites in protein-coding sequence to achieve sub-optimal expression. These substitutions only involve the ORFS, and thus non-protein-coding genome regions are not affected. Also, CPD involves only synonymous codon substitutions, and thus amino acid coding is unaffected. In addition, CPD applied to one or several genes typically involves hundreds or thousands of nucleotide changes, and thus should be highly refractory to de-attenuation. Recently, CPD was applied to poliovirus and influenza virus and was shown to result in attenuated strains. We designed the following set of four CPD RSV genomes in which the indicated ORFs were recoded: (i) Min A; NS1, NS2, N, P, M, and SH (i.e., the left-hand third of the genome); (ii) Min B; G and F (located in the middle of the genome); (iii) Min L; L (located at the right-hand end of the genome); and (iv) Min FLC; all ORFs except M2-1 and M2-2. The recoded genome regions were synthesized commercially and the four CPD viruses were constructed and recovered by reverse genetics. All of the CPD viruses were temperature-sensitive (level of sensitivity: Min FLC>Min L>Min B>Min A) for replication in vitro. We speculate that CDP may slow down the rate of translation sufficiently to create protein-folding problems that are exacerbated by increased temperature. All of the CPD mutants grew less efficiently in vitro than wild type (wt) RSV, even at the permissive temperature of 32C (growth efficiency: wt>Min L>Min A>Min FLC>Min B). Thus, CPD of G and F ORFs provided the greatest effect. The CPD viruses exhibited a range of restriction in mice and African Green Monkeys (AGM) and induced immunity against wt RSV. This study identified new vaccine candidates for RSV and showed that CPD of a nonsegmented negative-strand RNA virus can rapidly generate vaccine candidates with a range of attenuation phenotypes. We used gene-deletion HMPV strains to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on HMPV uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro, and on subsequent MDDC maturation and activation of autologous T cells. Deletion of G and SH (delSHG) conferred increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4+ T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4+ T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4+ T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to ∆SHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4+ T cells. This study describes a previously unknown mechanism of virus immune evasion. This is of interest because reinfection by HMPV is common throughout life without need for significant antigenic change, suggesting that protective immune response to HMPV is incomplete and short-lived. The present study provides a mechanism that might contribute to suppressing host immunity.