Influence of Mixed Retrovirus Infections on Leukemia and Neurological disease

用外源亲嗜性鼠白血病病毒（MuLV）感染小鼠或在某些小鼠品系中表达内源亲嗜性MuLV导致产生通过与内源逆转录病毒序列重组而衍生的宿主范围变体。纯化总是涉及用内源性多变性逆转录病毒的env基因取代外源性病毒的受体结合区。 这些变体利用不同的细胞表面受体进行感染，并且与仅感染鼠细胞的亲嗜性MuLV相比，能够感染来自几个物种的细胞;因此命名为多嗜性MuLV。 MuLVs在小鼠中诱导的许多疾病都涉及多方MuLVs的参与。 这些包括诱导增殖、免疫和神经系统疾病。 我们早期的研究表明，亲嗜性和多变性MuLV在宿主中的相互作用在促进发病机制中起着重要作用。 这在通过外源性逆转录病毒与宿主的内源性逆转录病毒重组产生的嗜亲性和多变性MuLV的混合感染中以及通过接种嗜亲性和多变性MuLV的混合物产生的混合感染中是明显的。在后一种情况下，用不同性质的逆转录病毒接种小鼠，深刻地影响了病毒的复制和感染的结果。 这些包括一种多变性MuLV诱导增殖性疾病的高度显著延迟，以及另一种多变性分离株导致神经系统疾病突然发展的显著协同效应。 在这两种情况下，共接种小鼠中的嗜多性病毒载量显著增加，而嗜亲性MuLV水平不变。 此外，在共接种小鼠中，嗜亲性MuLV在嗜亲性病毒粒子内几乎完全假型化。 有许多可能的机制，可以促进深刻的体内扩增的多变MuLV。 这些可能包括由于嗜亲性病毒粒子内的假型化或共感染细胞中的嗜多性病毒的反式激活而增强的病毒传播。为了在一个不太复杂的系统中研究这些问题，我们将这些研究扩展到研究体外细胞系的混合逆转录病毒感染。 亲嗜性和多嗜性混合感染的体外研究反映了许多体内观察结果。 嗜多性基因组在嗜亲性病毒粒子内高度假型化，并且与仅用嗜多性MuLV感染的细胞相比，嗜多性病毒的复制高度升高。 此外，嗜亲性感染性保持不变。 最近的研究表明，在慢性感染的细胞中，感染性多变性MuLV的扩增是不均匀的。我们已经发现，感染的培养物由细胞的异质混合物组成，其中一些释放高水平的感染性病毒，而另一些仅释放痕量或不可检测水平的感染性多变性MuLV。 在用亲嗜性病毒感染克隆后，即使那些仅释放痕量或不可检测水平的感染性的克隆，多变感染性也增加到高水平。 在2016年，我们继续研究了用多变性MuLV感染的单个克隆细胞系。 对释放的病毒体的特异性感染性的评估揭示了与病毒体中Env蛋白水平相关的巨大差异。 仅释放痕量水平感染性的细胞系释放几乎没有Env SU蛋白的缺陷病毒体。 SU的缺乏通过用亲嗜性MuLV的超感染来补偿，所述亲嗜性MuLV提供Env蛋白并在超感染后增加含有Fr 98的MuLV的特异性感染性。 这种缺陷也可以通过用编码整个Fr 98基因组的Fr 98-RFP再感染来避免，这表明该缺陷不是克隆细胞系本身不能正确组装多变Env蛋白。 我们的研究结果表明，一个独特的背景下，最初的多变性病毒感染导致的状态，导致在细胞中的Env蛋白水平的抑制，并排除了有效的掺入到后代病毒粒子的多变性Env蛋白。 这可导致非生产性状态，其特征在于释放含有逆转录病毒RNA转录物的非感染性病毒体。 这种非生产性状态是可逆的重叠感染与第二个逆转录病毒，因此代表了一个潜伏的逆转录病毒感染不同的潜伏期，其特征在于转录沉默。在这些实验过程中，我们观察到我们的一些克隆系的感染性病毒产量下降。其中一个克隆在长时间传代后没有释放可检测到的感染性颗粒，但对亲嗜性F-MuLV的重复感染或Fr 98-RFP的再感染有反应。 这表明感染性病毒沉默的一种渐进的和可能受控的机制，其可能在体内逆转录病毒感染以及内源性逆转录病毒的表达中具有重要作用。

Infection of mice with exogenous ecotropic murine leukemia viruses (MuLVs) or expression of endogenous ecotropic MuLVs in certain mouse strains results in the generation of host-range variants derived by recombination with endogenous retroviral sequences. Recombination invariably involves the substitution of the receptor binding region of the exogenous virus with the env gene of endogenous polytropic retroviruses. These variants utilize a different cell surface receptor for infection and are capable of infecting cells from several species compared to ecotropic MuLVs that only infect murine cells; hence the designation, polytropic MuLVs. The induction of many diseases in mice by MuLVs involves the participation of polytropic MuLVs. These include the induction of proliferative, immunological and neurological disorders. Our earlier studies indicate that the interactions of ecotropic and polytropic MuLVs in the host play a major role in facilitating pathogenesis. This is apparent in mixed infections of ecotropic and polytropic MuLVs generated by recombination of exogenous retroviruses with endogenous retroviruses of the host, as well as in mixed infections generated by inoculation of mixtures of ecotropic and polytropic MuLVs. In the latter case inoculation of mice with retroviruses of different properties profoundly affects the replication of the viruses and the outcome of the infection. These included a highly significant delay in the induction of proliferative disease with one polytropic MuLV and a profound synergistic effect resulting in the abrupt development of a neurological disease with another polytropic isolate. In both instances the polytropic virus load in the co-inoculated mice was markedly enhanced while the level of the ecotropic MuLV was unchanged. Furthermore, the polytropic MuLV was nearly completely pseudotyped within ecotropic virions in co-inoculated mice. There are a number of possible mechanisms that could facilitate the profound in vivo amplification of the polytropic MuLVs. These may include enhanced spread of the virus due to pseudotyping within ecotropic virions or possibly transactivation of the polytropic virus in co-infected cells. To examine these questions in a less complex system we extended these studies to examine mixed retrovirus infections of an in vitro cell line. In vitro studies of ecotropic and polytropic mixed infections mirror many of the in vivo observations. The polytropic genome is highly pseudotyped within ecotropic virions and the replication of the polytropic virus is highly elevated compared to cells infected with the polytropic MuLV alone. Furthermore, the ecotropic infectivity remains unaltered. Recent studies indicate that the amplification of infectious polytropic MuLV in chronically infected cells is not uniform. We have found that the infected cultures are comprised of a heterogeneous mixture of cells, some of which release high levels of infectious viruses and some which release only trace or undetectable levels of infectious polytropic MuLV. Upon infection of the clones with an ecotropic virus, polytropic infectivity increased to high levels even with those clones that released only trace or undetectable levels of infectivity. In 2016 we have continued our investigations of individual clonal cell lines infected with the polytropic MuLV. Assessment of the specific infectivitys of the released virions revealed large differences that correlate with the level of the Env protein in the virions. A cell line that released only trace levels of infectivity released defective virions that were nearly devoid of the Env SU protein. The deficiency in SU is compensated by superinfection with an ecotropic MuLV which provides the Env proteins and increases the specific infectivity of Fr98-containing MuLVs upon superinfection. This deficiency is also circumvented by reinfection with a Fr98-RFP that encodes the entire Fr98 genome indicating that the deficiency in not an inability of the clonal cell line itself to properly assemble the polytropic Env protein. Our results suggest a unique context of the initial polytropic virus infection leading to a state that results in the suppression of Env protein levels in the cell and precludes the efficient incorporation the polytropic Env protein into progeny virions. This can result in a non-productive state characterized by the release of non-infectious virions containing retroviral RNA transcripts. This non-productive state is reversible by superinfection with a second retrovirus and thus represents a latent retroviral infection distinct from latency characterized by transcriptional silencing. We have observed decreases in the infectious virus production by some of our clonal lines during the course of these experiments. One of the clones after prolonged passage releases no detectable infectious particles, yet responds to superinfection by the ecotropic F-MuLV or reinfection by Fr98-RFP. This suggests a progressive and perhaps controlled mechanism of infectious virus silencing that may have important roles in in vivo retroviral infections as well as in the expression of endogenous retroviruses.