A Viral Non‐Coding Region Determining Neuropathogenicity of Murine Leukemia Virus A8 Is Responsible for Envelope Protein Expression in the Rat Brain

Abstract

A neuropathogenic variant of the Friend murine leukemia virus (Fr-MLV), clone A8, induces spongiform neurodegeneration without inflammatory infiltrates when infected into neonatal rats (15). Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 (10) identified the env gene of A8 as a primary determinant for the induction of spongiform neurodegeneration in the brains of infected rats (15). Several MLVs induce spongiform neurodegeneration when infected into neonatal mice and/or rats (1,2,4,6,17,19,20). One common feature of these viruses is that the primary determinant for the induction of neurodegenerative disease in the central nervous system (CNS) is the env gene. Some uninfected neurons exhibit cytopathogenicity in the brains of rats infected with neuropathogenic viruses, indicating an indirect mechanism for MLV-induced neuropathogenicity (11,18). It has been suggested that the uncleaved Env precursor protein, or Env protein with differential processing of N-linked sugar, is correlated with retrovirus-induced spongiform neurodegeneration (3,7,8,13,14). However, the pathomechanism of spongiosis induced by Env is still not understood. We recently reported that a 0.3-kb fragment containing the R-U5-5' leader sequence of A8 is necessary for neuropathogenicity in addition to the env gene (16). The 0.3-kb fragment seems to influence Env protein expression in cultured cells. Immunohistochemical studies of infected rat brains support this view, but quantitative and qualitative analysis of Env protein expressed in the brain has not been carried out yet. In this study, we detected the Env protein in rat brains infected with neuropathogenic and non-neuropathogenic viruses by Western blot. We also evaluated processing of the Env protein and its expression level by comparing it with Gag. Furthermore, we compared the ratios of Env/Gag expression between infected brains and spleens.R7f and Rec5, chimeras of neuropathogenic A8 and non-neuropathogenic 57 viruses, were inoculated into newborn Lewis rats intraperitoneally and/or intracerebrally, as described previously (15, 16). The animals were kept according to the guidelines determined by the committee at our university. Genomic DNA was isolated from the brains and spleens of rats infected with R7f and Rec5 using QIAGEN Genomic-tips (QIAGEN) according to the manufacturer's instructions. PCR to detect viral DNA was performed as described previously A Viral Non-Coding Region Determining Neuropathogenicity of Murine Leukemia Virus A8 Is Responsible for Envelope Protein Expression in the Rat BrainSayaka Takase-Yoden*, Misaho Wada, and Rihito Watanabe Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan Received August 15, 2005; in revised form, October 26, 2005. Accepted December 6, 2005 Abstract: Friend murine leukemia virus clone A8 causes spongiform neurodegeneration in the rat brain. A 0.3-kb fragment containing the R-U5-5' leader sequence of A8 is required in addition to the A8-env ...

The 0.3-kb fragment containing the R-U5-5’leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA

“…Between the A8 and 57 sequences within the 0.3-kb fragment, 17 nucleotides differ (Figure 1). In our previous study, neuropathogenic R7f, which contains the A8-0.3-kb fragment and the A8- env gene on the background of 57, was shown to increase Env expression about 3-fold compared to non-neuropathogenic Rec5, which contains the 57-0.3-kb fragment and the A8- env gene on the background of 57 [5,6]. To investigate the function of the 0.3-kb fragment in viral gene expression, the full-length viral genomes of Rec5 and R7f were recombined with the luc gene, in which the viral env gene was replaced with the luc gene to produce the luciferase expression vectors Rec5-L and R7f-L, respectively (Figure 2A).…”

“…Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing the R-U5-5’leader sequence as an important determinant of neuropathogenicity, in addition to the env gene of A8 as the primary determinant [5]. The A8-Env protein expression level is also correlated with neuropathogenicity [5,6]. Chimeric virus Rec5, which contains the A8- env gene on the background of 57, did not exhibit neuropathogenicity.…”

“…In contrast, the chimeric virus R7f, which contains a 0.3-kb fragment of A8 and the A8- env gene on the background of 57, induced spongiform neurodegeneration. It has been shown that the expression level of Env protein in both R7f-infected cultured cells and in brains of R7f-infected rats was higher than in the Rec5-infected cultured cells and brains of Rec5-infected rats [5,6]. These findings suggested that the 0.3-kb fragment influences Env protein expression.…”

Kinetic studies of the effect of a 17‐nucleotide difference in the 0.3‐kb region containing the R‐U5‐5′ leader sequence of Friend murine leukemia virus on viral gene expression

“…We also previously showed that, in addition to the env gene, a 0.3-kb fragment containing the R-U5-5 0 leader sequence of Fr-MLV A8 was necessary for neuropathogenicity (32). The 0.3-kb fragment influences the amount of Env protein in cultured cells and in rat brains (32,33). This fragment contains functional domains, such as a signal for poly(A) addition to mRNA that works in the 3 0 LTR, a PBS for reverse transcription, and a 5 0 splice site.…”

“…Alternatively, cellular effectors, such as cytokines and inducible nitric oxide synthase, may be aberrantly expressed in the brains of infected animals (24)(25)(26)(27)(28)(29)(30)(31). In a previous study of the viral clone A8, we showed that a high level of expression of A8-Env protein in rat brain was correlated with neuropathogenicity (32)(33)(34). We also previously showed that, in addition to the env gene, a 0.3-kb fragment containing the R-U5-5 0 leader sequence of Fr-MLV A8 was necessary for neuropathogenicity (32).…”