Inhibition of diverse opportunistic viruses by structurally optimized retrograde trafficking inhibitors

Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread. IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread.

Section: Methodsmentioning

confidence: 99%

Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread

Streck

Zhao

Sundstrom

et al. 2020

“…The virions produced in roller bottles were concentrated by ultracentrifugation on a 20% sorbitol cushion at 20,000 rpm for 1 h at 20°C in a Beckman SW32 rotor. HCMV stocks were titrated by serial dilution on MRC-5 cells and quantified by the immunological detection of immediate early proteins as previously described (67), using an antibody that detects the HCMV major immediate early proteins (68). Images of stained monolayers were acquired on a Nikon Eclipse Ti inverted microscope, and fluorescent nuclei were quantified using NIS Elements software.…”

Section: Methodsmentioning

confidence: 99%

Human Cytomegalovirus Decreases Major Histocompatibility Complex Class II by Regulating Class II Transactivator Transcript Levels in a Myeloid Cell Line

Sandhu

Buchkovich

2020

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that encodes many proteins to modulate the host immune response. Extensive efforts have led to the elucidation of multiple strategies employed by HCMV to effectively block NK cell targeting of virus-infected cells and the major histocompatibility complex (MHC) class I-primed CD8+ T cell response. However, viral regulation of the MHC class II-mediated CD4+ T cell response is understudied in endogenous MHC class II-expressing cells, largely because the popular cell culture systems utilized for studying HCMV do not endogenously express MHC class II. Of the many cell types infected by HCMV in the host, myeloid cells, such as monocytes, are of particular importance due to their role in latency and subsequent dissemination throughout the host. We investigated the impact of HCMV infection on MHC class II in Kasumi-3 cells, a myeloid-progenitor cell line that endogenously expresses the MHC class II gene, HLA-DR. We observed a significant reduction in the expression of surface and total HLA-DR at 72 h postinfection (hpi) and 120 hpi in infected cells. The decrease in HLA-DR expression was independent of the expression of previously described viral genes that regulate the MHC class II complex or the unique short (US) region of HCMV, a region expressing many immunomodulatory genes. The altered surface level of HLA-DR was not a result of increased endocytosis and degradation but was a result of a reduction in HLA-DR transcripts due to a decrease in the expression of the class II transactivator (CIITA). IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic herpesvirus that is asymptomatic for healthy individuals but that can lead to severe pathology in patients with congenital infections and immunosuppressed patients. Thus, it is important to understand the modulation of the immune response by HCMV, which is understudied in the context of endogenous MHC class II regulation. Using Kasumi-3 cells as a myeloid progenitor cell model endogenously expressing MHC class II (HLA-DR), this study shows that HCMV decreases the expression of HLA-DR in infected cells by reducing the transcription of HLA-DR transcripts early during infection independently of the expression of previously implicated genes. This is an important finding, as it highlights a mechanism of immune evasion utilized by HCMV to decrease the expression of MHC class II in a relevant cell system that endogenously expresses the MHC class II complex.

“…Virus was collected from cells and supernatant, and virions were concentrated by ultracentrifugation on a 20% sorbitol cushion at 20,000 rpm for 1 h at 20°C in a Beckman SW32 rotor. Titers were determined by serial dilutions on MRC-5 cells and were quantified by the immunological detection of immediate early proteins using the monoclonal D4 antibody against IE proteins (33). Images of stained monolayers were acquired on a Nikon Eclipse Ti inverted microscope, and fluorescent nuclei were quantified using NIS Elements software.…”

Section: Methodsmentioning

confidence: 99%

“…An antibody against a UL88 peptide was generated by GenScript. Generation of monoclonal antibodies against IE1/IE2 and UL71 was previously described (31,33). Commercial antibodies against pp28 (5C3; Virusys), CMV gB (2F12; Virusys), tubulin (DM1A; Millipore Sigma), GM130 (BD Biosciences), beta-actin (BA3R; Thermo Scientific), EEA1 (F.43.1; Thermo Scientific), and FLAG M2 (Millipore Sigma) were purchased.…”

Section: Methodsmentioning

confidence: 99%

UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument

Kumar

Cruz

Sandhu

et al. 2020

Self Cite

Little is known about the human cytomegalovirus (HCMV) tegument protein UL88. Large-scale genomic studies have reported disparate results for UL88-null viruses, reporting both no phenotype and a >1-log decrease in virus titers. UL88 has also been reported to interact with UL69 and UL48, but the functional relevance of this interaction is unknown. Here, we report that UL88, which is conserved among different viral strains, is dispensable for production of infectious HCMV virions in multiple HCMV strains and cell types. However, the specific infectivity of HCMV virions suffers in the absence of UL88, as more genomes are required per PFU. This may be a result of altered virion tegument protein composition, as Western blot analysis shows a significant reduction in the tegument levels of pp71, UL47, and UL48 in viruses lacking UL88. While an interaction between UL88 and UL48 has previously been reported, we show that UL88 can interact with UL47; however, UL88 does not appear to be part of a stable complex consisting of UL47 and UL48. These findings identify an important role for UL88 in incorporating the viral proteins UL47 and UL48 into the virion tegument layer. IMPORTANCE A better understanding of the role and functions of tegument proteins in HCMV, many of which remain uncharacterized, will contribute to our understanding of the biology of HCMV. The virus has a large genome, greater than 230 kb, and functional annotation of these genes is important for identifying novel targets for improving therapeutic intervention. This study identifies a role for a viral tegument protein with unknown function, UL88, in maintaining the proper tegument composition of HCMV virions. Virions produced in the absence of UL88 exhibit decreased fitness and require more genomes per infectious unit.