Research: Human cytomegalovirus replication and pathogenesis

Cytomegaloviruses are members of the herpesvirus 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 over 200 open reading frames with the potential to encode a protein.

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, 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.