Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul;89(13):6536-50.
doi: 10.1128/JVI.00284-15. Epub 2015 Apr 15.

Human Cytomegalovirus UL135 and UL136 Genes Are Required for Postentry Tropism in Endothelial Cells

Farah Bughio  1 , Mahadevaiah Umashankar  2 , Jean Wilson  3 , Felicia Goodrum  4
Affiliations

Human Cytomegalovirus UL135 and UL136 Genes Are Required for Postentry Tropism in Endothelial Cells

Farah Bughio et al. J Virol. 2015 Jul.

Abstract

Endothelial cells (ECs) are a critical target of viruses, and infection of the endothelium represents a defining point in viral pathogenesis. Human cytomegalovirus (HCMV), the prototypical betaherpesvirus, encodes proteins specialized for entry into ECs and delivery of the genome to the nuclei of ECs. Virus strains competent to enter ECs replicate with differing efficiencies, suggesting that the virus encodes genes for postentry tropism in ECs. We previously reported a specific requirement for the UL133/8 locus of HCMV for replication in ECs. The UL133/8 locus harbors four genes: UL133, UL135, UL136, and UL138. In this study, we find that while UL133 and UL138 are dispensable for replication in ECs, both UL135 and UL136 are important. These genes are not required for virus entry or the expression of viral genes. The phenotypes associated with disruption of either gene reflect phenotypes observed for the UL133/8NULL virus, which lacks the entire UL133/8 locus, but are largely distinct from one another. Viruses lacking UL135 fail to properly envelop capsids in the cytoplasm, produce fewer dense bodies (DB) than the wild-type (WT) virus, and are unable to incorporate viral products into multivesicular bodies (MVB). Viruses lacking UL136 also fail to properly envelop virions and produce larger dense bodies than the WT virus. Our results indicate roles for the UL135 and UL136 proteins in commandeering host membrane-trafficking pathways for virus maturation. UL135 and UL136 represent the first HCMV genes crucial for early- to late-stage tropism in ECs.

Importance: Human cytomegalovirus (HCMV) persists in the majority of the world's population. While typically asymptomatic in healthy hosts, HCMV can cause significant morbidity and mortality in immunocompromised or naïve individuals, particularly transplant patients and patients with congenital infections, respectively. Lifelong persistence of the virus may also contribute to age-related pathologies, such as vascular disease. One aspect of HCMV infection contributing to complex and varied pathogenesis is the diverse array of cell types that this virus infects in the host. The vascular endothelium is a particularly important target of infection, contributing to viral dissemination and likely leading to CMV complications following transplantation. In this work, we identify two viral gene products required for postentry tropism in endothelial cells. Identifying tropism factors required for replication in critical cell targets of infection is important for the development of strategies to restrict virus replication.

PubMed Disclaimer

Figures

FIG 1
FIG 1
UL135 and UL136 are required for replication in endothelial cells. (A) HMVEC were infected with the TB40/E-WT, -UL133/8NULL, -UL133STOP, -UL135STOP, -UL136GalK, or -UL138STOP virus at an MOI of 0.05. (B) HMVEC were infected with the TB40/E-WT, -UL136NULL, or -UL135STOP/UL138STOP virus at an MOI of 0.05. Virus yields were measured in lysates harvested over a time course (measured in days postinfection) by TCID50 on fibroblasts. The values plotted are averages for 3 to 4 independent experiments. The standard deviation for each time point is indicated by error bars.
FIG 2
FIG 2
UL135 and UL136 exhibit unique distributions throughout the cytoplasm of infected cells. HMVEC were infected at an MOI of 2 with one of two TB40/E recombinant viruses (UL135Myc or UL136Myc), each expressing the indicated protein with a C-terminal Myc epitope tag. At 144 hpi, cells were processed for indirect immunofluorescence microscopy using monoclonal antibodies specific to the Golgi marker GM130 and the Myc epitope tag. Nuclei were stained by DAPI. UL135 is located primarily at the plasma membrane (arrowheads) and is dispersed in the cytoplasm, whereas UL136 colocalizes extensively with the Golgi marker (arrows). Bars, 10 μm.
FIG 3
FIG 3
Accumulation of viral genomes and transcripts in infected HMVEC. (A) Total DNA was isolated from HMVEC infected with TB40/E-WT (filled bars), -UL135STOP (shaded bars), or -UL136GalK (open bars) (MOI, 0.1) at the time points indicated. Genomes were quantified by real-time PCR using primers specific to UL99, and values were normalized to those for the cellular gene RNaseP. The results of one experiment representative of three total experiments are shown. (B to D) Transcripts for UL123/IE1 (B), UL69 (C), and UL32/pp150 (D) were quantified from cDNA synthesized from total RNA isolated from infected HMVEC over a time course. Transcript levels were normalized to those for β-actin. The bars represent average values from three independent experiments. ΔCT, change in threshold cycle.
FIG 4
FIG 4
Accumulation of IE, early, and late viral proteins in infected HMVEC. (A) HMVEC were infected at an MOI of 2 with TB40/E-WT, -UL135STOP, or -UL136GalK. Protein lysates were harvested over the time course indicated (in hours postinfection [hpi]) and were analyzed by immunoblotting using antibodies specific to the 72-kDa IE1 and 86-kDa IE2 proteins, the early protein UL44, the late protein pp28, and β-actin. β-Actin served as a loading control. (B and C) Quantification of the average IE1 (B) and pp28 (C) protein levels (normalized to those for actin) from three independent experiments. The standard deviations are indicated. Student's t test indicates that there is no significant difference between WT and mutant viruses at each time point.
FIG 5
FIG 5
UL135 and UL136 are required for efficient formation of the viral assembly compartment. HMVEC were infected at an MOI of 2 with TB40/E-WT, -UL133STOP, -UL135STOP, UL136GalK, or UL138STOP. At 144 h postinfection, cells were processed for indirect immunofluorescence microscopy using monoclonal antibodies specific to the Golgi marker GM130 and the late viral protein pp28. Nuclei were stained by DAPI. Both the UL135STOP and UL136GalK viruses failed to reorganize the intracellular membranes into the VAC. A merge of all three images is shown at the right. Bars, 10 μM.
FIG 6
FIG 6
UL135 and UL136 are required for virion maturation. HMVEC were infected at an MOI of 4 with the TB40/E-WT (A), -UL135STOP (B), or -UL136GalK (C) virus. At 5 days postinfection, cells were fixed, embedded, and sectioned for TEM. Representative micrographs are shown to illustrate the accumulation and maturation of virus particles in the cytoplasm. Three distinct virus particle morphologies are observed: normal virions with characteristic HCMV morphology (filled arrowheads), aberrantly enveloped virions (open arrowheads), and nonenveloped capsids (filled arrows). DB, dense body. Bars, 500 nm. (D) Seven hundred to 1,300 total virions were counted in 30 to 45 cells. The percentages of normal virions (open portions of bars), aberrantly enveloped virions (shaded portions of bars), and nonenveloped capsids (filled portions of bars) in each infection are illustrated.
FIG 7
FIG 7
UL135 and UL136 mutant virus progeny in fibroblasts. MRC-5 fibroblasts were infected with the TB40/E-WT (A), -UL135STOP (B), or –UL136GalK (C) virus at an MOI of 4. At 5 days postinfection, cells were fixed, embedded, and sectioned for TEM. Representative micrographs are shown to illustrate the accumulation and maturation of virus particles in the cytoplasm. Three distinct virus particle morphologies are observed: normal virions with characteristic HCMV morphology (filled arrowheads), aberrantly enveloped virions (open arrowheads), and nonenveloped capsids (filled arrows). Bars, 2 μm.
FIG 8
FIG 8
Dense body formation impacted by the disruption of UL135 or UL136. HMVEC infected with the TB40/E-WT (A), -UL135STOP (B), or -UL136GalK (C) virus were fixed, embedded, and sectioned for TEM at 5 dpi. Representative micrographs are shown to illustrate the accumulation of DBs (arrows). Bars, 2 μm.
FIG 9
FIG 9
Enlarged DBs are not due to increased accumulation of DB proteins. (A) HMVEC were infected at an MOI of 2 with the TB40/E-WT, -UL135STOP, or -UL136GalK virus. Protein lysates were harvested over the time course indicated (in hours postinfection [hpi]) and were analyzed by immunoblotting using antibodies specific to the pp150 and pp65 proteins. β-Actin served as a loading control. (B and C) Quantification of the average pp65 (B) and pp150 (C) protein levels (normalized to actin) in three independent experiments. The standard deviations are indicated. Student's t test indicated that there was no significant difference between WT and mutant viruses at each time point.
FIG 10
FIG 10
pUL135 is required for the incorporation of viral products into MVB. HMVEC were infected at an MOI of 4 with the TB40/E-WT (A), -UL135STOP (B), or UL136GalK (C) virus. At 5 days postinfection, cells were fixed, embedded, and sectioned for TEM. Representative micrographs are shown to illustrate the accumulation of MVB (arrows) and the incorporation of virions (arrowheads) and DBs (open arrowheads) into MVB. Bars, 500 nm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Britt W. 2008. Manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease. Curr Top Microbiol Immunol 325:417–470. doi:10.1007/978-3-540-77349-8_23. - DOI - PubMed
    1. Goodrum F, Caviness K, Zagallo P. 2012. Human cytomegalovirus persistence. Cell Microbiol 14:644–655. doi:10.1111/j.1462-5822.2012.01774.x. - DOI - PMC - PubMed
    1. Jarvis MA, Nelson JA. 2007. Molecular basis of persistence and latency, chapter 42 In Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, Yamanishi K (ed), Human herpesviruses: biology, therapy, and immunoprophylaxis. Cambridge University Press, Cambridge, United Kingdom. - PubMed
    1. Boeckh M, Geballe AP. 2011. Cytomegalovirus: pathogen, paradigm, and puzzle. J Clin Invest 121:1673–1680. doi:10.1172/JCI45449. - DOI - PMC - PubMed
    1. Mocarski ES, Shenk T, Pass RF. 2007. Cytomegaloviruses, p 2701–2673. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (ed), Fields virology, 5th ed Lippincott Williams & Wilkins, Philadelphia, PA.

Publication types

Substances

LinkOut - more resources