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Thomas Shenk

Human cytomegalovirus replication and pathogenesis

Cytomegaloviruses are members of the herpes virus family. Human cytomegalovirus (HCMV) infections are widespread and subclinical in the vast majority of cases, but the virus exhibits increased virulence in the very young and old and in immunocompromised individuals. Some infants develop perceptual defects as a result of cytomegalovirus infection. Transplant recipients, cancer patients, and AIDS patients, all of whom exhibit decreased immune function, suffer a variety of clinical manifestations resulting from cytomegalovirus infection, including mononucleosis and pneumonia. The HCMV particle carries a viral genome comprised of linear double-stranded DNA. Its DNA is the largest of all known human viruses, and it includes 227 open reading frames, each with the potential to encode a protein of at least 100 amino acids.

We employ two approaches to explore mechanisms underlying HCMV replication and pathogenesis. To investigate the functions of viral genes, we study the biochemical activities of individual viral genes or we create mutant viruses lacking specific viral genes and examine the nature of their growth defect. To explore the molecular basis of pathogenesis, we utilize DNA arrays to monitor the expression of both viral and cellular genes within infected cells.

We have focused our functional analysis of the viral genome on genes that encode proteins that are localized to the tegument domain of the virus particle. The tegument is comprised of about 25 proteins that are delivered to the newly infected cell together with the viral DNA, and they have the opportunity to function at a very early stage in the infectious process. We chose several of these proteins for study because we anticipated that they might exhibit critical regulatory functions that prepare the cell for infection and initiate the program of viral gene expression. We have discovered that two of these proteins regulate cell cycle progression. The UL69 tegument protein blocks cell cycle progression late in the G1 compartment. Presumably, the block in late G1 provides the virus with an environment for replication in which cellular gene products required for replication have been induced, and the cell does not compete for these products since it is not licensed to begin replication of its own DNA. We are now exploring the mechanism by which UL69 protein blocks cell cycle progression. In addition to blocking cell cycle progression at the G1/S boundary, HCMV is known to stimulate quiescent cells to move from G0 into the G1 compartment of the cell cycle. Then further progression is blocked by UL69. We have shown that this stimulatory activity is carried out by a second tegument protein, pp71. We are now exploring the mechanism by which pp71 activates quiescent cells.

We have employed DNA array technology to identify changes in cellular gene expression that could be important for viral replication, represent a cellular response to infection, or contribute to pathogenesis. We monitored the levels of mRNAs corresponding to 6,500 cellular cDNAs at various times after infection, and found that the steady-state level of 258 mRNAs changed by a factor of four or more after infection. We are now exploring the physiological relevance of several of the changes we have cataloged. For example, multiple components of the pathway producing prostaglandins, including cyclooxygenase 2, are induced in infected cells. Since prostaglandins are proinflammatory, we anticipated that activation of this pathway might be an antiviral response of cells to infection. Surprisingly, however, high doses of cyclooxygenase 2 inhibitors markedly inhibit viral replication. Activation of this pathway facilitates viral replication, and we are now working to identify the point in the HCMV replication cycle at which cyclooxygenase 2 activity is needed.

We have also constructed an array that contains DNAs corresponding to HCMV open reading frames with the potential to encode proteins of at least 100 amino acids. We had noted that virions contain RNA, so we used the array to analyze RNA isolated from purified, ribonuclease A-treated virions. Interestingly, five virus-coded mRNAs that are synthesized with late kinetics are packaged within virions. This is the first demonstration that a herpesvirus particle contains mRNAs. Presumably, these RNAs are packaged so that they can be translated very early within a newly infected cell, even before the viral DNA reaches the nucleus and begins to be transcribed. We are creating mutant viruses to explore the functions of the virion RNAs. 

Selected Publications

Moorman NJ, Sharon-Friling R, Shenk T, Cristea IM. (2009) A targeted spatial-temporal proteomic approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation. Mol Cell Proteomics. [Epub ahead of print]

Mitchell DP, Savaryn JP, Moorman NJ, Shenk T, Terhune SS. (2009) Human cytomegalovirus UL28 and UL29 open reading frames encode a spliced mRNA and stimulate accumulation of immediate-early RNAs. J Virol. 83: 10187-10197. PubMed

Lilja AE, Shenk T. (2008) Efficient replication of rhesus cytomegalovirus variants in multiple rhesus and human cell types. Proc Natl Acad Sci 105: 19950-19955. PubMed

Schröer J, Shenk T. (2008) Inhibition of cyclooxygenase activity blocks cell-to-cell spread of human cytomegalovirus. Proc Natl Acad Sci. 105: 19468-19473. PubMed

Munger J, Bennett BD, Parikh A, Feng XJ, McArdle J, Rabitz HA, Shenk T, Rabinowitz JD. (2008) Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol. 26: 1179-1186. PubMed

Cuevas-Bennett C, Shenk T. (2008) Dynamic histone H3 acetylation and methylation at human cytomegalovirus promoters during replication in fibroblasts. J Virol. 82: 9525-9536. PubMed

Murphy E, Shenk T. (2008) Human cytomegalovirus genome. Curr Top Microbiol Immunol. 325: 1-19. PubMed

Moorman NJ, Cristea IM, Terhune SS, Rout MP, Chait BT, Shenk T. (2008) Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Cell Host Microbe. 3: 253-262. PubMed

Lilja AE, William Chang WL, Barry PA, Becerra SP, Shenk TE. (2007) Functional genetic analysis of Rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor. J Virol. 82: 2170-2181. PubMed

Wang D, Yu QC, Schröer J, Murphy E, Shenk T. (2007) Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells. Proc Natl Acad Sci USA 104: 20037-20042. PubMed

Terhune S, Torigoi E, Moorman N, Silva M, Qian Z, Shenk T, Yu D (2007). Human Cytomegalovirus UL38 protein blocks apoptosis. J Virol 81: 3109-3123. PubMed

Weinberger LS, Shenk T (2006). An HIV feedback resistor: auto-regulatory circuit deactivator and noise buffer. PLoS Biol 5: e9. PubMed

Sharon-Friling R, Goodhouse J, Colberg-Poley AM, Shenk T (2006). Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores. Proc Natl Acad Sci 103: 19117-19122. PubMed

Kulesza CA, Shenk T (2006). Murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse. Proc Natl Acad Sc.103: 18302-18307. PubMed

Feng X, Schroer J, Yu D, Shenk T (2006). Human Cytomegalovirus pUS24 is a virion protein that functions very early in the replication cycle. J Virol 80: 8371-8378. PubMed

Munger J, Yu D, Shenk T (2006). UL26-deficient human cytomegalovirus produces virions with hypophosphorylated pp28 tegument protein that is unstable within newly infected cells. J Virol 80: 3541-3548. PubMed

Gaspar M and Shenk T (2006). Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins. Proc Natl Acad Sci USA 103: 2821-2826 . PubMed

Wang D and Shenk T (2005). Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci USA 102: 18153-18158. PubMed

Wang D and Shenk T (2005). Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism. J Virol 79: 10330-10338. PubMed

Silva MC, Schroer J and Shenk T (2005). Human cytomegalovirus cell-to-cell spread in the absence of an essential assembly protein. Proc Natl Acad Sci USA 102: 2081-2086. PubMed

Wang D, Bresnahan W and Shenk T (2004). Human cytomegalovirus encodes a highly specific RANTES decoy receptor. Proc Natl Acad Sci USA 101: 16642-16647. PubMed

Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Pasa-Tolic L, Wang D, Camp DG 2nd, Rodland K, Wiley S, Britt W, Shenk T, Smith RD, Nelson JA (2004). Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J Virol 78: 10960-10966. PubMed

Terhune SS, Schroer J and Shenk T (2004). RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration. J Virol 78: 10390-10398. PubMed

Nevels M, Paulus C and Shenk T (2004). Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation. Proc Natl Acad Sci USA 101: 17234-17239. PubMed

Nevels M, Brune W and Shenk T (2004). SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication. J Virol 78: 7803-7812. PubMed

Kulesza CA and Shenk T (2004). Human cytomegalovirus 5-kilobase immediate-early RNA is a stable intron. J Virol 78: 13182-13189. PubMed

Goodrum F, Jordan CT, Terhune SS, High K and Shenk T (2004). Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations. Blood 104: 687-695. PubMed

Challacombe JF, Rechtsteiner A, Gottardo R, Rocha LM, Browne EP, Shenk T, Altherr MR, Brettin TS (2004). Evaluation of the host transcriptional response to human cytomegalovirus infection. Physiol Genomics 18: 51-62. PubMed

Adamo JE, Schroer J and Shenk T (2004). Human cytomegalovirus TRS1 protein is required for efficient assembly of DNA-containing capsids. J Virol 78: 10221-10229. PubMed