Margo H. Furman

The Human Cytomegalovirus US10 Gene Product Delays Trafficking of Major Histocompatibility Complex Class I Molecules

ABSTRACT Human cytomegalovirus (HCMV) US10 encodes a glycoprotein that binds to major histocompatibility complex (MHC) class I heavy chains. While expression of US10 delays the normal trafficking of MHC class I molecules out of the endoplasmic reticulum, US10 does not obviously facilitate or inhibit the action of two other HCMV-encoded MHC class I binding proteins, US2 and US11.

Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ERin vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chainsin vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.

Lessons from viral manipulation of protein disposal pathways.

Detection and elimination of viral pathogens is a continual challenge for all organisms, even mammals which benefit from a sophisticated immune system. Many classes of viruses are resistant to complete elimination by the immune system and persist in a latent or minimally replicative state for the lifetime of the host. This persistence is often without clinical consequence to the host, as extensive damage would compromise survival of the pathogen. Examination of viral gene products has revealed a multitude of strategies employed by viruses to escape from innate and adaptive host defense mechanisms. These features enable long-term infection and create a delicate balance between the virus’s ability to replicate and spread and the host’s ability to control these events. Although viruses replicate inside host cells, their presence is not concealed from the watchful eye of the immune system. Numerous cellular mechanisms provide protection where mechanical and humoral defenses cannot. NK cells are among the first lymphocytes to sense the release of IFN-α and IFN-β, as well as perturbations in expression of MHC class I molecules and other surface molecules, all of which are triggered by viral invasion of cells. Several days after exposure to the virus, specific CTLs become activated. These CTLs, recognizing viral antigenic peptides bound to host cell MHC class I molecules, then begin to eliminate infected cells by releasing cytolytic and proapoptotic factors like perforin and granzyme, IFN-γ, TNF-α, and TNF-β (1). This system is essential to root out intracellular pathogens, as demonstrated by the increased susceptibility of animals lacking CTLs to many types of pathogens, particularly viruses (2). The MHC class I pathway provides numerous points of interference for viral pathogens. This is logical, as MHC class I proteins continually sample the intracellular space inhabited by viruses and present this intracellular material on the surface of host cells for recognition by CTLs. The remarkable variety of proteins encoded by viruses to avoid CTL recognition shows that this arm of the adaptive immune system must be disabled or circumvented by successful intracellular pathogens. At the same time, the sheer diversity of molecular mechanisms by which CTL avoidance is achieved makes it difficult to predict how newly identified immune-evasive molecules accomplish this end (see refs. 3, 4 for recent reviews). Here, we discuss new data and unresolved questions in this exciting field.

The ubiquitin-proteasome pathway in thymocyte apoptosis: caspase-dependent processing of the deubiquitinating enzyme USP7 (HAUSP).

Programmed cell death (apoptosis) is crucial for thymocyte development. We analyzed the role of the ubiquitin (Ub)-proteasome pathway in dexamethasone-triggered and TCR-mediated apoptosis in fetal thymic organ culture (FTOC). Proteasome activity was increased in apoptotic thymocytes, as visualized by active-site labeling of proteasomal beta subunits. The activity of deubiquitinating enzymes in murine apoptotic thymocytes was likewise examined by active-site labeling. We show that the deubiquitinating enzyme USP7 (HAUSP) is proteolytically processed upon dexamethasone-, gamma-irradiation-, and antigen-induced cell death. Such processing of HAUSP does not occur in caspase 3-/- thymocytes, or upon pretreatment of wild type thymocytes with the general caspase inhibitor ZVAD-fmk. Thus, our results suggest that thymocyte apoptosis leads to modification of deubiquitinating enzymes by caspase activity and may provide an additional link between the ubiquitin-proteasome pathway and the caspase cascade during programmed cell death.

Can viruses help us to understand and classify the MHC class I molecules at the maternal-fetal interface?

Placental expression of HLA-C, HLA-E, and HLA-G locus products is now well described. However, to date, the functional relevance of these MHC class I products at the maternal-fetal interface is incompletely described. We propose here that HLA-C, -E, and -G comprise a distinct and cohesive group of MHC class I products. This hypothesis is supported by a growing body of data, including that obtained through the study of viral immune evasion. Continued investigation of viral interactions with MHC class I products promises to help us to define those specific attributes of HLA-C, -E, and -G that define their common characteristics.