Felicia Goodrum Sterling

Our research focuses on human cytomegalovirus (CMV) latency. CMV is a beta herpesvirus that infects the majority of the population worldwide and is the leading cause of infectious-disease related birth defects. CMV replicates in a variety of cell types, but its genome is uniquely maintained without the production of progeny in hematopoietic cells. This latent state enables the virus to persist for a lifetime in healthy individuals without causing disease. However, reactivation of CMV from latency can cause life-threatening disease in immunocompromised patients such as stem cell and solid organ transplant recipients and AIDS patients. In spite of its critical importance to the infectious cycle and pathogenesis, the mechanisms underlying CMV latency have remained obscure. Latency is a delicate balance dependent on complex interactions between the virus and the host at the molecular, cellular, and systemic levels. Primary impediments to understanding CMV latency have been the lack of suitable in vitro and in vivo models for CMV latency. Recently, we have developed a novel in vitro system for studying hematopoietic progenitor cells giving a great deal of attention to hematopoietic cell biology. Using this system, we have identified a hematopoietic progenitor subpopulation (CD34+/CD38-) that uniquely supports a latent infection when infected in vitro. Our work seeks to identify and characterize viral and cellular determinants of CMV latency using this system. Clinical strains of CMV, but not highly-passaged laboratory-adapted strains, can establish a latent infection in hematopoietic cells infected in vitro. Interestingly, laboratory-adapted strains are missing a 15 kilobase segment of DNA (termed the ULb’ region) that is found in all clinical strains. From these findings, we hypothesize that viral determinants exist that contribute to the latent infection in hematopoietic progenitor cells. We have identified a 5 kilobase sequence, and more specifically the UL138 open reading frame (ORF), within the ULb’ region unique to clinical strains that promotes a latent infection in hematopoietic cells. Currently, our focus is to determine the mechanism by which viral determinants function in promoting latency using molecular, genetic, and biochemical approaches. We are also interested in developing an in vivo model for studying CMV infection and latency in human hematopoietic cells. We propose to develop a physiologically relevant mouse model using immunocompromised mice engineered to support human hematopoiesis. The mouse will serve as a sophisticated incubator, providing a microenvironment for differentiation of CMV-infected human hematopoietic cells that cannot be recapitulated in culture systems. This mouse model will allow us to analyze complex interactions between CMV-infected hematopoietic cells and their microenvironment as they pertain to CMV latency. Using this system, we will determine the effects of CMV infection on hematopoietic reconstitution following transplantation and the ability of infected progenitor cells to disseminate latently infected cells through differentiation. Our studies will define key mechanisms governing CMV latency and identify molecular targets for improved antiviral treatments to prevent CMV disease. Elucidating the mechanisms underlying CMV latency is critical to controlling CMV disease in immunocompromised patients and to prevent congenital infection.
An Endothelial Cell Specific Requirement For The Ul133 Ul138 Locus Of Human Cytomegalovirus For Efficient Virus Maturation. Source: Journal Of Virology
Human Cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well-defined, the molecular determinants regulating post-entry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb' region of the HCMV genome as important for the latent infection in CD34+ hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or FIX HCMV strains. ECs infected with a virus lacking the entire locus, UL133-UL138(NULL), complete the immediate early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138(NULL) exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138(NULL)-infected ECs, Golgi stacks were disrupted and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138(NULL)-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138(NULL), suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type dependent, post-entry tropism determinant required for viral assembly.<br /><br />
Interactions Between Proteins Encoded Within The Human Cytomegalovirus Ul133 Ul138 Locus. Source: Journal Of Virology
June 6th, 2012 PMID: 22674978 Felicia Goodrum Sterling
We previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.<br /><br />
Human Cytomegalovirus Persistence. Source: Cellular Microbiology
March 8th, 2012 PMID: 22329758 Felicia Goodrum Sterling
Viral persistence is the rule following infection with all herpesviruses. The β-herpesvirus, human cytomegalovirus (HCMV), persists through chronic and latent states of infection. Both of these states of infection contribute to HCMV persistence and to the high HCMV seroprevalence worldwide. The chronic infection is poorly defined molecularly, but clinically manifests as low-level virus shedding over extended periods of time and often in the absence of symptoms. Latency requires long-term maintenance of viral genomes in a reversibly quiescent state in the immunocompetent host. In this review, we focus on recent advances in the biology of HCMV persistence, particularly with respect to the latent mode of persistence. Latently infected individuals harbour HCMV genomes in haematopoietic cells and maintain large subsets of HCMV-specific T-cells. In the last few years, impressive advances have been made in understanding virus-host interactions important to HCMV infection, many of which will profoundly impact HCMV persistence. We discuss these advances and their known or potential impact on viral latency. As herpesviruses are met with similar challenges in achieving latency and often employ conserved strategies to persist, we discuss current and future directions of HCMV persistence in the context of the greater body of knowledge regarding α- and γ-herpesviruses persistence.<br /><br />
A Novel Human Cytomegalovirus Locus Modulates Cell Type Specific Outcomes Of Infection. Source: P Lo S Pathogens
December 29th, 2011 PMID: 22241980 Felicia Goodrum Sterling
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 IL2Rγ(c) (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.<br /><br />
Stress Inducible Alternative Translation Initiation Of Human Cytomegalovirus Latency Protein P Ul138. Source: Journal Of Virology
June 30th, 2010 PMID: 20592073 Felicia Goodrum Sterling
We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3' coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3' end of each transcript and is preceded by an extensive 5' sequence (approximately 0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5' ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5' of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.<br /><br />
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