Influence of Mixed Retrovirus Infections on Leukemia and Neurological disease

小鼠感染外源性生态型小鼠白血病病毒(MuLV)或内源性生态型MULV在某些品系中的表达导致通过与内源性逆转录病毒序列重组而产生宿主范围变异。重组总是涉及用内源性多嗜性逆转录病毒的env基因替换外源病毒的受体结合区。这些变异株利用不同的细胞表面受体进行感染，与仅感染小鼠细胞的生态型MuLV相比，它们能够感染几个物种的细胞；因此被命名为多向性MuLV。MULV诱导小鼠多种疾病的发生涉及多向MULV的参与。这包括导致增殖性、免疫性和神经性疾病。我们早期的研究表明，宿主中生态型和多向性MuLV之间的相互作用在促进发病过程中发挥了重要作用。这在外源性逆转录病毒与宿主内源性逆转录病毒重组产生的生态型和多嗜型MuLV的混合感染中以及在接种生态型和多嗜型MuLV的混合感染中表现得很明显。在后一种情况下，给小鼠接种不同性质的逆转录病毒会对病毒的复制和感染的结果产生深远的影响。这些包括一种多嗜性MuLV在引起增殖性疾病方面的非常显著的延迟，以及一种深刻的协同效应，导致与另一种多嗜性分离株突然发展成一种神经系统疾病。在这两种情况下，联合接种的小鼠的多嗜性病毒载量显着增加，而生态型MuLV的水平没有变化。此外，在联合接种的小鼠中，多嗜性MuLV几乎完全是生态型病毒粒子内的假型。 有许多可能的机制可以促进多向性MuLV在体内的深刻扩增。这些可能包括由于生态型病毒粒子内的伪分型或可能在共同感染的细胞中多嗜性病毒的反式激活而加强病毒的传播。为了在一个不那么复杂的系统中检查这些问题，我们将这些研究扩展到检查体外细胞系的混合逆转录病毒感染。生态型和多源性混合感染的体外研究反映了体内的许多观察结果。在生态型病毒粒子中，多角体基因组高度假型，与仅感染多角体MuLV的细胞相比，多角体病毒的复制高度增强。此外，生态型传染性保持不变。 最近的研究表明，传染性多角体小鼠白血病病毒在慢性感染细胞中的扩增并不均匀。我们发现，受感染的培养物是由不同种类的细胞组成的，其中一些细胞释放高水平的传染性病毒，而另一些细胞只释放微量或检测不到的传染性多角体病毒。一旦克隆感染了生态型病毒，即使是那些只释放微量或无法检测到的传染性水平的克隆，多向传染性也会增加到高水平。 2016年，我们继续对感染多嗜性MuLV的单个克隆细胞系进行了研究。对释放的病毒粒子的特定感染力的评估显示，与病毒粒子中环境蛋白的水平相关的差异很大。一种只释放微量传染性的细胞系释放了几乎没有Env Su蛋白的有缺陷的病毒粒子。SU的缺乏可以通过重叠感染生态亲水性小鼠白血病病毒来弥补，这种病毒提供了环境蛋白，并在重叠感染时增加了含有Fr98的小病毒的特异性感染力。这种缺陷也可以通过再次感染编码整个Fr98基因组的Fr98-RFP来避免，这表明这种缺陷并不是克隆细胞系本身无法正确组装多变的Env蛋白。 我们的结果表明，最初的多嗜性病毒感染具有独特的背景，导致细胞中的Env蛋白水平受到抑制，并排除了将多嗜性Env蛋白有效地整合到后代病毒粒子中的可能性。这可能导致以释放含有逆转录病毒RNA转录本的非传染性病毒粒子为特征的非生产状态。这种非生产状态可通过重叠感染第二种逆转录病毒而逆转，因此是一种潜伏的逆转录病毒感染，不同于以转录沉默为特征的潜伏期。我们观察到，在这些实验过程中，我们的一些克隆系减少了传染性病毒的产生。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，但对生态型F-MuLV的重叠感染或Fr98-RFP的再感染有反应。这提示了一种渐进的、可能受控制的感染性病毒沉默机制，这种机制可能在体内逆转录病毒感染和内源性逆转录病毒的表达中发挥重要作用。

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.