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We previously reported that a (CTG)n expansion causes spinocerebellar ataxia type 8 (SCA8), a slowly progressive ataxia with reduced penetrance. We now report a transgenic mouse model in which the full-length human SCA8 mutation is transcribed using its endogenous promoter. (CTG)116 expansion, but not (CTG)11 control lines, develop a progressive neurological phenotype with in vivo imaging showing reduced cerebellar-cortical inhibition. 1C2-positive intranuclear inclusions in cerebellar Purkinje and brainstem neurons in SCA8 expansion mice and human SCA8 autopsy tissue result from translation of a polyglutamine protein, encoded on a previously unidentified antiparallel transcript (ataxin 8, ATXN8) spanning the repeat in the CAG direction. The neurological phenotype in SCA8 BAC expansion but not BAC control lines demonstrates the pathogenicity of the (CTG-CAG)n expansion. Moreover, the expression of noncoding (CUG)n expansion transcripts (ataxin 8 opposite strand, ATXN8OS) and the discovery of intranuclear polyglutamine inclusions suggests SCA8 pathogenesis involves toxic gain-of-function mechanisms at both the protein and RNA levels.

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Retroviral Gag proteins perform important functions in viral assembly, but are also involved in other steps in the viral life cycle. Conventional mutational analysis has yielded considerable information about domains essential for these functions, yet many regions of gag remain uncharacterized. We used genetic footprinting, a technique that permits the generation and simultaneous analysis of large numbers of mutations, to perform a near-saturation mutagenesis and functional analysis of 639 nucleotides in the gag region of Moloney murine leukemia virus. We report here the resulting functional map defined by eight footprints representing regions of Moloney murine leukemia virus gag , some previously uncharacterized, that are essential for replication. We found that significant portions of matrix and p12 proteins were tolerant of insertions, in contrast to the N-terminal half of capsid, which was not. We analyzed 30 mutants from our library by using conventional methods to validate the footprints. Six of these mutants were characterized in detail, identifying the precise stage at which their replication is blocked. In addition to providing the most comprehensive functional map of a retroviral gag gene, our study demonstrates the abundance of information that can be gleaned by genetic footprinting of viral sequences.

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans., Author Summary Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection and causes developmental abnormalities, including hearing loss and developmental delay. Although there is no therapy for congenital HCMV disease, there is evidence from both human and animal studies that antibodies can have efficacy in this setting. Such studies have focused exclusively on polyclonal antibodies, in which the targets of protective antibodies are unknown. Guinea pigs have been used as a model of human maternal fetal transmission of infection because of similarities in placental anatomy between human and guinea pig. Furthermore, guinea pig CMV (GPCMV) has been demonstrated to cross the placenta and cause fetal infection and loss, similar to the effects of infection with HCMV. However, the kinetics of maternal and fetal infection in this model has not been carefully investigated. In this work, we have delineated the kinetics of maternal to fetal infection and found that congenital infection is rapid following maternal infection. Importantly, we demonstrate that a monoclonal antibody against a protein critical for viral entry protects pregnant guinea pigs against fetal infection. Thus, our studies may be informative for development of a therapeutic intervention to treat congenital HCMV infection in humans.

The primary sequence of the long unique region L-DNA (L for low GC) of rhesus monkey rhadinovirus (RRV) isolate 26-95 was determined. The L-DNA consists of 130,733 bp that contain 84 open reading frames (ORFs). The overall organization of the RRV26-95 genome was found to be very similar to that of human Kaposi sarcoma-associated herpesvirus (KSHV). BLAST search analysis revealed that in almost all cases RRV26-95 coding sequences have a greater degree of similarity to corresponding KSHV sequences than to other herpesviruses. All of the ORFs present in KSHV have at least one homologue in RRV26-95 except K3 and K5 (bovine herpesvirus-4 immediate-early protein homologues), K7 (nut-1), and K12 (Kaposin). RRV26-95 contains one MIP-1 and eight interferon regulatory factor (vIRF) homologues compared to three MIP-1 and four vIRF homologues in KSHV. All homologues are correspondingly located in KSHV and RRV with the exception of dihydrofolate reductase (DHFR). DHFR is correspondingly located near the left end of the genome in RRV26-95 and herpesvirus saimiri (HVS), but in KSHV the DHFR gene is displaced 16,069 nucleotides in a rightward direction in the genome. DHFR is also unusual in that the RRV26-95 DHFR more closely resembles HVS DHFR (74% similarity) than KSHV DHFR (55% similarity). Of the 84 ORFs in RRV26-95, 83 contain sequences similar to the recently determined sequences of the independent RRV isolate 17577. RRV26-95 and RRV17577 sequences differ in that ORF 67.5 sequences contained in RRV26-95 were not found in RRV17577. In addition, ORF 4 is significantly shorter in RRV26-95 than was reported for RRV17577 (395 versus 645 amino acids). Only four of the corresponding ORFs between RRV26-95 and RRV17577 exhibited less than 95% sequence identity: glycoproteins H and L, uracil DNA glucosidase, and a tegument protein (ORF 67). Both RRV26-95 and RRV17577 have unique ORFs between positions 21444 to 21752 and 110910 to 114899 in a rightward direction and from positions 116524 to 111082 in a leftward direction that are not found in KSHV. Our analysis indicates that RRV26-95 and RRV17577 are clearly independent isolates of the same virus species and that both are closely related in structural organization and overall sequence to KSHV. The availability of detailed sequence information, the ability to grow RRV lytically in cell culture, and the ability to infect monkeys experimentally with RRV will facilitate the construction of mutant strains of virus for evaluating the contribution of individual genes to biological properties.

All members of the Herpesviridae family contain sequences for a highly conserved glycoprotein B (gB) gene. We investigated the phylogenetic relationships of gB sequences from eight independent rhadinovirus isolates obtained from three species: rhesus ( Macaca mulatta ), cynomologus ( Macaca fasicularis ), and pig-tailed ( Macaca nemestrina ) macaques. Samples were derived from monkeys housed at four separate facilities. Analysis of these eight independent gB sequences revealed five regions of heterogeneity within the 823- to 829-amino-acid polypeptides: residues 1 to 65, 120 to 185, 255 to 300, 352 to 393, and 412 to 457. The remaining regions of gB were highly conserved among the different macaque isolates. Overall divergence among these gene sequences ranged from 0.1 to 7.2% at the amino acid level. Phylogenetic trees constructed with our macaque rhadinovirus gB sequences and those derived from additional subfamilies or genera (alpha, beta, gamma-1, and gamma-2) revealed that the macaque gB sequences branched with other gamma-2 herpesvirus gB sequences and that within the gamma-2 genera, the macaque gB sequences clustered as a distinct branch. The eight macaque rhadinovirus gB sequences were all approximately equidistant from Kaposi sarcoma-associated herpesvirus (KSHV) gB sequences and had a shorter evolutionary distance to KSHV gB sequences than to any other herpesvirus, including the gamma-2 herpesvirus saimiri (HVS) of New World squirrel monkeys. The macaque gB sequences did not cluster according to the facility of origin, but did cluster according to the species of origin, displaying less intraspecies divergence (0.1 to 2.9%) than interspecies divergence (3.3 to 7.2%). These results demonstrate a close relatedness of rhadinovirus isolates from different macaque species.

Unique among primates, the colobine monkeys have adapted to a predominantly leaf-eating diet by evolving a foregut that utilizes bacterial fermentation to breakdown and absorb nutrients from such a food source. It has been hypothesized that pancreatic ribonuclease (pRNase) has been recruited to perform a role as a digestive enzyme in foregut fermenters, such as artiodactyl ruminants and the colobines. We present molecular analyses of 23 pRNase gene sequences generated from 8 primate taxa, including 2 African and 2 Asian colobine species. The pRNase gene is single copy in all noncolobine primate species assayed but has duplicated more than once in both the African and Asian colobine monkeys. Phylogenetic reconstructions show that the pRNase-coding and noncoding regions are under different evolutionary constraints, with high levels of concerted evolution among gene duplicates occurring predominantly in the noncoding regions. Our data suggest that 2 functionally distinct pRNases have been selected for in the colobine monkeys, with one group adapting to the role of a digestive enzyme by evolving at an increased rate with loss of positive charge, namely arginine residues. Conclusions relating our data to general hypotheses of evolution following gene duplication are discussed.

Direct sequencing of mitochondrial DNA (mtDNA) following amplification using the polymerase chain reaction (PCR) [1] has found widespread use in population genetic and phylogenetic research over the past few years. Recently, nuclear copies of mitochondrial genes have been reported in diverse eukaryotic species, often confounding such research (reviewed in [2,3]). Under certain circumstances, nuclear pseudogenes can be amplified more efficiently than the intended mtDNA target, even when using as template mtDNA that has been purified by gradient centrifugation [4]. If the transfer of the gene copy to the nucleus happened recently, it can be difficult – if not impossible – to identify the legitimate mitochondrial sequence. Here, we present a simple method that can identify expressed mitochondrial genes, using the cytochrome b gene of the particularly problematical proboscis monkey as an example. Because mtDNA is transcribed and processed into polyadenylated mRNAs [5,6], reverse transcription coupled to PCR can be used to amplify the expressed mitochondrial version. This method produced an unambiguous sequence for the proboscis monkey mitochondrial cytochrome b gene; in contrast, traditional DNA-based PCR methods produced ambiguous sequence, because many nuclear pseudogenes were present. Phylogenetic analysis of the cytochrome b gene suggests that the proboscis monkey groups with the Asian langurs, rather than forming a sister taxon to all Asian and African colobines as was previously suggested [7]. Reverse transcriptase-coupled PCR should be applicable to many other cases of nuclear transfer of mtDNA, including those involving ribosomal genes.

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.

Factors accounting for long-term nonprogression may include infection with an attenuated strain of human immunodeficiency virus type 1 (HIV-1), genetic polymorphisms in the host, and virus-specific immune responses. In this study, we examined eight individuals with nonprogressing or slowly progressing HIV-1 infection, none of whom were homozygous for host-specific polymorphisms (CCR5-Δ32,CCR2-64I, andSDF-1-3′A) which have been associated with slower disease progression. HIV-1 was recovered from seven of the eight, and recovered virus was used for sequencing the full-length HIV-1 genome; full-length HIV-1 genome sequences from the eighth were determined following amplification of viral sequences directly from peripheral blood mononuclear cells (PBMC). Longitudinal studies of one individual with HIV-1 that consistently exhibited a slow/low growth phenotype revealed a single amino acid deletion in a conserved region of the gp41 transmembrane protein that was not seen in any of 131 envelope sequences in the Los Alamos HIV-1 sequence database. Genetic analysis also revealed that five of the eight individuals harbored HIV-1 with unusual 1- or 2-amino-acid deletions in the Gag sequence compared to subgroup B Gag consensus sequences. These deletions in Gag have either never been observed previously or are extremely rare in the database. Three individuals had deletions in Nef, and one had a 4-amino-acid insertion in Vpu. The unusual polymorphisms in Gag, Env, and Nef described here were also found in stored PBMC samples taken 3 to 11 years prior to, or in one case 4 years subsequent to, the time of sampling for the original sequencing. In all, seven of the eight individuals exhibited one or more unusual polymorphisms; a total of 13 unusual polymorphisms were documented in these seven individuals. These polymorphisms may have been present from the time of initial infection or may have appeared in response to immune surveillance or other selective pressures. Our results indicate that unusual, difficult-to-revert polymorphisms in HIV-1 can be found associated with slow progression or nonprogression in a majority of such cases.