The translation of the unspliced and partially spliced viral mRNAs that encode the late, structural proteins of HIV-1 depends on the viral-protein Rev. Oligomeric binding of Rev to the Rev response element (RRE) in these mRNAs promotes their export from the nucleus and thus controls their expression. Here, we compared the effects of hydrophobic to hydrophilic mutations within the oligomerization domain of Rev using assays for oligomeric RNA binding, protein structure, and export from the nucleus. Oligomeric RNA binding alone does not correlate well with RNA transport activity in the subset of mutants. However, protein structure as judged by CD spectroscopy does correlate well with Rev function. The oligomeric assembly of Rev-L18T is impaired but exhibits minor defects in structure and retains a basal level of activity in vivo. The prevalence of L18T in infected individuals suggests a positive selection mechanism for L18T modulation of Rev activity that may delay the onset of AIDS.Keywords: HIV-1; Rev; RRE; nuclear export; viral latencyThe Rev protein of HIV-1 controls gene expression by promoting the nuclear export of unspliced and partially spliced viral mRNA (Malim et al. 1989b;Pollard and Malim 1998;Hope 1999). Rev recognizes these mRNAs through both the binding of an arginine-rich RNA binding domain (RBD) to a specific binding site within the Rev response element (RRE) (Malim et al. 1989a;Bogerd and Greene 1993) and the self-association of Rev monomers into higher-order oligomeric complexes on the RRE (Malim et al. 1989a;Bogerd and Greene 1993). It is this strong tendency to self-associate that has thwarted attempts to obtain structural information of full-length Rev at the atomic level. Understanding the essential contribution made by Rev oligomerization to influence the course of the viral infection (Malim and Cullen 1991;Madore et al. 1994;Mann et al. 1994) has been hampered by the resulting lack of detailed structural information on the oligomeric complexes. Many questions concerning the connection between Rev structure and oligomeric Rev-RRE assembly (Blanco et al. 2001;Havlin et al. 2007), the self-association tendency of Rev in the absence of its cognate RRE (Wingfield et al. 1991;Cole et al. 1993;Havlin et al. 2007), and the observation of Rev variants with

CDR3 regions containing two D segments, or containing the footprints of VH replacement events, have been reported in both mice and humans. However, the 12–23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for VH replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential VH replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the “cryptic heptamer” but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and VH replacement are rare occurrences in the mouse.

A major component in controlling V(D)J recombination is differential accessibility through localized changes in chromatin structure. Attachment of DNA to the nuclear matrix via matrix attachment region (MAR) sequences, and interaction with MAR-binding proteins have been shown to alter chromatin conformation, promote histone acetylation, and influence gene transcription. In this study, the flanking regions of several human and mouse Ig VH and Ig Vκ genes were analyzed extensively for the presence of MARs by in vitro matrix-binding assay, and for interaction with the MAR-binding proteins cut-like protein x/CCAAT-displacement protein (Cux/CDP), B cell regulator of IgH transcription (Bright), and special AT-rich sequence-binding protein (SATB1) by EMSA. Cux/CDP and SATB1 are associated with repression, while Bright is an activator of Ig transcription. Binding sites were identified in the vicinity of all analyzed Ig V genes, and were also found flanking TCR Vβ genes. We also show that the binding sites of the different factors do not always occur at MAR sequences. MAR sequences were also found within the Ig V loci at a much higher frequency than throughout the rest of the genome. Overall, the frequency and location of binding sites relative to the coding regions, and the strength of DNA-protein interaction showed much heterogeneity. Thus, variations in factor binding and MAR activity could potentially influence the extent of localized accessibility to V(D)J recombination and thus could play a role in unequal rearrangement of individual V genes. These sites could also contribute to effective transcription of Ig genes in mature and/or activated B cells, bringing both the promoter as well as the enhancer regions into close proximity at the nuclear matrix.

Lymphocyte infiltration of salivary and lacrimal glands leading to diminished secretion and gland destruction as a result of apoptosis is thought to be pivotal in the pathogenesis of Sjögren's syndrome (SS). The cytoskeletal protein alpha-fodrin is cleaved during this apoptotic process, and a strong antibody (Ab) response is elicited to a 120-kd fragment of cleaved alpha-fodrin in the majority of SS patients, but generally not in other diseases in which apoptosis also occurs. Little is known about the anti-alpha-fodrin autoantibody response on a molecular level. To address this issue, IgG phage display libraries were generated from the bone marrow of two SS donors and a panel of anti-alpha-fodrin IgGs was isolated by selection on alpha-fodrin immunoblots. All of the human monoclonal Abs (hmAbs) reacted with a 150-kd fragment and not with the 120-kd fragment or intact alpha-fodrin, indicating that the epitope recognized became exposed after alpha-fodrin cleavage. Analysis of a large panel of SS patients (defined by the strict San Diego diagnostic criteria) showed that 25% of SS sera exhibited this 150-kd alpha-fodrin specificity. The hmAbs stained human cultured salivary acinar cells and the staining was redistributed to surface blebs during apoptosis. They also stained inflamed acinar/ductal epithelial cells in SS salivary tissue biopsies, and only partially co-localized with monoclonal Abs recognizing the full-length alpha-fodrin. Our study shows that in SS patients, neoepitopes on the 150-kd cleaved product of alpha-fodrin become exposed to the immune system, frequently eliciting anti-150-kd alpha-fodrin Abs in addition to the previously reported anti-120-kd Abs. The anti-150-kd alpha-fodrin hmAbs may serve as valuable reagents for the study of SS pathogenesis and diagnostic analyses of SS salivary gland tissue.

Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline DH gene in mice of the Ighj haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Ighj mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Ighj mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had ∼2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.