Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with Kd values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγc null-humanized mouse model. The UL133-UL138 NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations.

Human cytomegalovirus (HCMV) exists indefinitely in infected individuals by a yet poorly characterized latent infection in hematopoietic cells. We previously demonstrated a requirement for the putative UL138 open reading frame (ORF) in promoting a latent infection in CD34؉ hematopoietic progenitor cells (HPCs) infected in vitro. In our present study, we have identified two coterminal transcripts of 2.7 and 3.6 kb and a 21-kilodalton (kDa) protein (pUL138) that are derived from the UL138 locus with early-late gene kinetics during productive infection. The UL138 transcripts and protein are detected in both fibroblasts and HPCs. A recombinant virus, FIX-UL138 STOP , that synthesizes the UL138 transcripts but not the protein exhibited a partial loss-of-latency phenotype in HPCs, similar to the phenotype observed for the UL138-null recombinant virus. This finding suggests that the UL138 protein is required for latency, but it does not exclude the possibility that the UL138 transcripts or other ORFs also contribute to latency. The mechanisms by which pUL138 contributes to latency remain unknown. While the 86-and 72-kDa immediate-early proteins were not detected in HPCs infected with HCMV in vitro, pUL138 did not function directly to suppress expression from the major immediate-early promoter in reporter assays. Interestingly, pUL138 localizes to the Golgi apparatus in infected cells but is not incorporated into virus particles. The localization of pUL138 to the Golgi apparatus suggests that pUL138 contributes to HCMV latency by a novel mechanism. pUL138 is the first HCMV protein demonstrated to promote an infection with the hallmarks of latency in CD34 ؉ HPCs.

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling.

Ir-catalyzed borylations of 4-substituted benzonitriles are described. In contrast to electrophilic aromatic substitutions and directed ortho metalations, C-H activation/borylation enables functionalization at the 2-position, adjacent to the cyano group, when the 4-subsitutent is larger than cyano. When an excess of borane reagent is used, diborylation can be achieved with a single regioisomer being formed in certain cases. Extension of sterically directed borylation to cyano-substituted, five- and six-membered ring heterocycles is also reported.

The mechanisms by which viruses persist and particularly those by which viruses actively contribute to their own latency have been elusive. Here we report the existence of opposing functions encoded by genes within a polycistronic locus of the human cytomegalovirus (HCMV) genome that regulate cell type-dependent viral fates: replication and latency. The locus, referred to as the UL133-UL138 (UL133/8) locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. As part of the ULb= region of the genome, the UL133/8 locus is lost upon serial passage of clinical strains of HCMV in cultured fibroblasts and is therefore considered dispensable for replication in this context. Strikingly, we could not reconstitute infection in permissive fibroblasts from bacterial artificial chromosome clones of the HCMV genome where UL135 alone was disrupted. The loss of UL135 resulted in complex phenotypes and could ultimately be overcome by infection at high multiplicities. The requirement for UL135 but not the entire locus led us to hypothesize that another gene in this locus suppressed virus replication in the absence of UL135. The defect associated with the loss of UL135 was largely rescued by the additional disruption of the UL138 latency determinant, indicating a requirement for UL135 for virus replication when UL138 is expressed. In the CD34 ؉ hematopoietic progenitor model of latency, viruses lacking only UL135 were defective for viral genome amplification and reactivation. Taken together, these data indicate that UL135 and UL138 comprise a molecular switch whereby UL135 is required to overcome UL138-mediated suppression of virus replication to balance states of latency and reactivation. IMPORTANCEMechanisms by which viruses persist in their host remain one of the most poorly understood phenomena in virology. Herpesviruses, including HCMV, persist in an incurable, latent state that has profound implications for immunocompromised individuals, including transplant patients. Further, the latent coexistence of HCMV may increase the risk of age-related pathologies, including vascular disease. The key to controlling or eradicating HCMV lies in understanding the molecular basis for latency. In this work, we describe the complex interplay between two viral proteins, pUL135 and pUL138, which antagonize one another in infection to promote viral replication or latency, respectively. We previously described the role of pUL138 in suppressing virus replication for latency. Here we demonstrate a role of pUL135 in overcoming pUL138-mediated suppression for viral reactivation. From this work, we propose that pUL135 and pUL138 constitute a molecular switch balancing states of latency and reactivation.

Using a novel human organ donor tissue resource, Gordon et al. reveal how CMV-specific T cells are distributed and function in multiple sites in the context of viral persistence, revealing new insights into immune control of CMV in the body.

Human cytomegalovirus (HCMV) is a complex DNA virus with a 230-kb genome encoding 170 and up to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb= region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and, possibly, the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibited distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane-bound isoforms of UL136 differed between when they were expressed exogenously and when they were expressed in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection-specific factors. While UL136, like most ULb= genes, was dispensable for replication in fibroblasts, the soluble 23-and 19-kDa isoforms suppressed virus replication. In CD34؉ hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23-and 19-kDa isoforms resulted in increased replication and a loss of the latency phenotype, similar to the effects of the UL138 latency determinant encoded within the same genetic locus. Our work suggests a complex interplay between the UL136 isoforms which balances viral replication in multiple cell types and likely contributes to the cell typedependent phenotypes of the UL133/8 locus and the outcome of HCMV infection. IMPORTANCEHCMV is a significant cause of morbidity in immunocompromised individuals, including transplant patients. The lifelong persistence of the virus results in a high seroprevalence worldwide and may contribute to age-related pathologies, such as atherosclerosis. The mechanisms of viral persistence are poorly understood; however, understanding the molecular basis of persistence is imperative for the development of new treatments. In this work, we characterize a complex HCMV gene, UL136, which is expressed as five protein isoforms. These isoforms arise predominantly from complex transcriptional mechanisms, which contribute to an increased coding capacity of the virus. Further, the UL136 isoforms oppose the activity of one another to balance HCMV replication in multiple cell types. We identify soluble isoforms of UL136 that function to suppress virus replication in fibroblasts and in CD34 ؉ HPCs for latency.