Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells.

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34+ hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction and is required for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like UL135, the largest UL136 isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency, and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for replication. We generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the UL135 mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34+ HPCs and in humanized NOD-scid IL2Rγcnull (huNSG) mice. This finding suggests that while UL135 is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for UL135. Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135, whereby UL135 may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cells and humanized mouse models.

HCMV UL8 interaction with ß-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

CD34+ Hematopoietic Progenitor Cell Subsets Exhibit Differential Ability To Maintain Human Cytomegalovirus Latency and Persistence.

In human cytomegalovirus (HCMV)-seropositive patients, CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study, we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism.

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

Viral binding-induced signaling drives a unique and extended intracellular trafficking pattern during infection of primary monocytes.

We initiated experiments to examine the infection of monocytes postentry. New data show that human cytomegalovirus (HCMV) DNA is detected in the nucleus beginning only at 3 d postinfection in monocytes, compared with 30 min postinfection in fibroblasts and endothelial cells, suggesting that HCMV nuclear translocation in monocytes is distinct from that seen in other cell types. We now show that HCMV is initially retained in early endosomes and then moves sequentially to the trans-Golgi network (TGN) and recycling endosomes before nuclear translocation. HCMV is retained initially as a mature particle before deenvelopment in recycling endosomes. Disruption of the TGN significantly reduced nuclear translocation of viral DNA, and HCMV nuclear translocation in infected monocytes was observed only when correct gH/gL/UL128-131/integrin/c-Src signaling occurred. Taken together, our findings show that viral binding of the gH/gL/UL128-131 complex to integrins and the ensuing c-Src signaling drive a unique nuclear translocation pattern that promotes productive infection and avoids viral degradation, suggesting that it represents an additional viral evasion/survival strategy.

Viral and host network analysis of the human cytomegalovirus transcriptome in latency.

HCMV genes UL135 and UL138 play opposing roles regulating latency and reactivation in CD34+ human progenitor cells (HPCs). Using the THP-1 cell line model for latency and reactivation, we designed an RNA sequencing study to compare the transcriptional profile of HCMV infection in the presence and absence of these genes. The loss of UL138 results in elevated levels of viral gene expression and increased differentiation of cell populations that support HCMV gene expression and genome synthesis. The loss of UL135 results in diminished viral gene expression during an initial burst that occurs as latency is established and no expression of eleven viral genes from the ULb' region even following stimulation for differentiation and reactivation. Transcriptional network analysis revealed host transcription factors with potential to regulate the ULb' genes in coordination with pUL135. These results reveal roles for UL135 and UL138 in regulation of viral gene expression and potentially hematopoietic differentiation.

A novel human cytomegalovirus locus modulates cell type-specific outcomes of infection.

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.

IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin.

Human cytomegalovirus (HCMV) is linked to the acceleration of vascular diseases such as atherosclerosis and transplant vasculopathy. One of the hallmarks of these diseases is angiogenesis (AG) and neovessel formation. Endothelial cells (ECs) are an integral part of AG and are sites of HCMV persistence. AG requires multiple synchronous processes that include EC proliferation, migration, and vessel stabilization. Virus-free supernatant (secretome) from HCMV-infected ECs induces AG. To identify factor(s) involved in this process, we performed a human cytokine array. Several cytokines were significantly induced in the HCMV secretomes including interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor, and IL-8/CXCL8. Using in vitro AG assays, neutralization of IL-6 significantly reduced neovessel formation. Addition of the HCMV secretome to preformed vessels extended neovessel survival, but this effect was blocked by neutralization of IL-6. In these cells, IL-6 prevented apoptosis by blocking caspase-3 and -7 activation through the induction of survivin. Neutralization of IL-6 receptor on ECs abolished the ability of HCMV secretome to increase survivin expression and activated effector caspases. Moreover, survivin shRNA expression induced rapid regression of tubule capillary networks in ECs stimulated with HCMV secretome and activated effector caspases. These observations may explain how CMV accelerates vascular disease despite limited infection in tissues.

UL135 and UL136 Epistasis Controls Reactivation of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is beta herpesvirus that persists indefinitely in the human host through a protracted, latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34+ hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple protein isoforms with differential roles in latency and reactivation. Like UL135, the largest UL136 isoform, UL136p33, is required for reactivation from latency in hematopoietic cells. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for reactivation. To test this, we generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact replication of the UL135-mutant virus in fibroblasts. However, in the context of infection in hematopoietic cells, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34+ HPCs and in humanized NOD- scid IL2Rγ c null (NSG) mice. This finding suggests that while UL135 is essential for reactivation, it functions at steps preceding the accumulation of UL136p33 and that stabilized expression of UL136p33 largely overcomes the requirement for UL135 in reactivation. Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135 whereby UL135 may initiate events early in reactivation that will result in the accumulation of UL136p33 to a threshold required for productive reactivation. SIGNIFICANCE: Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a life-long latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immune compromised. Defining virus-host and virus-virus interactions important for HCMV latency, reactivation and replication is critical to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cell and humanized mouse models.

Late gene expression-deficient cytomegalovirus vectors elicit conventional T cells that do not protect against SIV.

Rhesus cytomegalovirus-based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory-based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71). Here, we examined the impact of an even more stringent attenuation strategy on vector-induced immune protection against SIV. Fusion of the FK506-binding protein (FKBP) degradation domain to Rh108, the orthologue of the essential human CMV (HCMV) late gene transcription factor UL79, generated RhCMV/SIV vectors that conditionally replicate only when the FK506 analog Shield-1 is present. Despite lacking in vivo dissemination and reduced innate and B cell responses to vaccination, Rh108-deficient 68-1 RhCMV/SIV vectors elicited high-frequency, durable, EM-biased, SIV-specific T cell responses in RhCMV-seropositive RMs at doses of ≥ 1 × 106 PFU. Strikingly, elicited CD8+ T cells exclusively targeted MHC-Ia-restricted epitopes and failed to protect against SIVmac239 challenge. Thus, Rh108-dependent late gene expression is required for both induction of MHC-E-restricted T cells and protection against SIV.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Advances in Model Systems for Human Cytomegalovirus Latency and Reactivation.

Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells. The study of HCMV latency has been hampered by difficulties in obtaining and culturing primary cells, as well as an inability to quantitatively measure reactivating virus, but recent advances in both in vitro and in vivo models of HCMV latency and reactivation have led to a greater understanding of the interplay between host and virus. Key differences in established model systems have also led to controversy surrounding the role of viral gene products in latency establishment, maintenance, and reactivation. This review will discuss the details and challenges of various models including hematopoietic progenitor cells, monocytes, cell lines, and humanized mice. We highlight the utility and functional differences between these models and the necessary experimental design required to define latency and reactivation, which will help to generate a more complete picture of HCMV infection of myeloid-lineage cells.

The interferon-inducible gene IFI16 secretome of endothelial cells drives the early steps of the inflammatory response.

The IFN-inducible human IFI16 gene is highly expressed in endothelial cells as well as epithelial and hematopoietic tissues. Previous gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 has revealed an increased expression of genes involved in inflammation and apoptosis. In this study, protein array analysis of the IFI16 secretome showed an increased production of chemokines, cytokines and adhesion molecules responsible for leukocyte chemotaxis. Functional analysis of the promoter for CCL20, the chemokine responsible for leukocyte recruitment in the early steps of inflammation, by site-specific mutation demonstrated that NF-κB is the main mediator of CCL20 induction at the transcriptional level. Finally, both Langerhans DC and B-lymphocyte migration triggered by supernatants from IFI16-overexpressing endothelial cells was partially inhibited by Ab inactivating CCL4, CCL5 and CCL20 chemokines. Altogether, these results demonstrate that the IFI16 gene, through its secretome, regulates proinflammatory activity of endothelial cells, thus corroborating its role in the early steps of inflammation.

The Elk-1 and serum response factor binding sites in the major immediate-early promoter of human cytomegalovirus are required for efficient viral replication in quiescent cells and compensate for inactivation of the NF-kappaB sites in proliferating cells.

The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate of IE gene transcription and thus affect the outcome of HCMV infection. We have shown previously that the NF-kappaB binding sites within the MIEP enhancer and cellular NF-kappaB activity induced by HCMV infection are required for efficient MIEP activity and viral replication in quiescent cells (P. Caposio, A. Luganini, G. Hahn, S. Landolfo, and G. Gribaudo, Cell. Microbiol. 9:2040-2054, 2007). We now show that the inactivation of either the Elk-1 or serum response factor (SRF) binding site within the enhancer also reduces MIEP activation and viral replication of recombinant HCMV viruses in quiescent fibroblasts. In these cells, we show that the expression of either Elk-1 or SRF is required for optimal IE gene expression, and that the HCMV-stimulated activation of the MEK1/2-ERK1/2 signaling axis leads to Elk-1 transcriptional competency. Furthermore, the replication kinetics of recombinant viruses in which NF-kappaB, Elk-1, and SRF binding sites all are inactivated demonstrate that the higher levels of Elk-1 and SRF binding to MIEP in proliferating cells can compensate even for a lack of HCMV-induced NF-kappaB-mediated MIEP transactivation. These observations highlight the importance of the combination of different MIEP binding sites to optimize IE gene expression in cells in different physiological states.

Development of a huBLT Mouse Model to Study HCMV Latency, Reactivation, and Immune Response.

Immunodeficient mice engrafted with human tissues provide a robust model for the in vivo investigation of human-restricted viruses such as human cytomegalovirus (HCMV). Several humanized mouse models have been developed and improved over the last 30 years. Here, we describe a protocol for the transplant of human hematopoietic stem cells with autologous fetal liver and thymic tissues into NOD.Cg-PrkdcscidIL2rγtm1Wjl mice to create a humanized bone marrow-liver-thymus model (huBLT) that can be infected with HCMV. The presence of human thymus allows the development of a functional human immune system, including HLA-restricted human T-cells and B-cells. Indeed, following infection, huBLT mice generate virus-specific CD4+ and CD8+ T-cell responses. Additionally, both HCMV-specific IgM and IgG B-cell responses can be detected. This huBLT model provides the first animal model to explore the adaptive human immune response to HCMV infection.