Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a widespread pathogen that causes an asymptomatic and latent infection in the majority of individuals. However, HCMV can also be life threatening for patients who are immunocompromised (e.g. patients with HIV, organ transplant recipients, or neonates). HCMV is the most common congenital infection and neonates may develop neurological abnormalities such as hearing or visual loss, and mental retardation. AIDS patients, cancer patients and transplant recipients may suffer various disorders resulting from HCMV infections including pneumonia, hepatitis, retinitis and encephalitis. Moreover, HCMV has also been associated to vascular diseases such as atherosclerosis, and recently to certain cancers.

We follow two approaches to investigate mechanisms underlying HCMV replication and pathogenesis. To identify cellular factors involved in HCMV replication, we evaluate viral production in knock-down cells and when virus yield reduction is observed we study the function of these cellular components in the virus life cycle. To investigate the molecular basis of HCMV pathogenesis, we study the function of HCMV-encoded chemokine receptors-like proteins.

We have focused our studies on HCMV replication on the processes that underlie virus envelopment and secretion. As an enveloped virus, HCMV replication is dependent on interaction with cellular membranes. Herpesvirus particles acquire their final envelopes from intracellular membranes prior to their secretion to the extracellular medium. We have recently found that HCMV acquires its final envelope from membranes that have characteristics of both the trans-Golgi network (TGN) and endosomes. Our recent data showed that HCMV induces the expression of cellular proteins involved in vesicle-mediated transport, including Rab27a - a small GTPase that regulates secretion of lysosomes-related organelles - and the SNARE protein Syntaxin3. These proteins associate in part with virus factories and silencing of these cellular components by RNA interference reduced HCMV production. These studies raised the exciting hypothesis that HCMV, and perhaps other herpesviruses, exploit the cellular machinery involved in lysosomes-related organelles function, for its envelopment and secretion. Understanding the molecular mechanisms at the interface of the virus-host interactions is crucial for the identification of novel targets for future antiviral strategies.

To test this hypothesis we adopt a multidisciplinary approach. The expression and localization of cellular components involved in lysosomes-realted organelles function is analyzed morphological (immunofluorescence and light microscopy; and immunogold labeling and electron microscopy) and biochemically in HCMV-infected cells. The interaction between these cellular components during viral infection is further characterized by co-immunoprecipitation assays. Their functional roles are investigated using RNAi and dominant negative mutants. RNAi specificity is also checked by reconstituting normal activity with codon-altered constructs that are resistant to the RNAi inhibition. The effect of silencing on virus replication is analyzed by infectivity assays to determine viral production and morphologically. These morphological studies include the analysis of the distribution of structural viral proteins in RNAi silencing cells by immunofluorescence and light microscopy together with the analysis of serial sections of HCMV-infected cell monolayers embedded in situ by electron microscopy.

This work will not only provide crucial new insights into the cellular basis of herpesvirus replication but also will shed light on basic aspects of cellular membrane biology. In this line, we have found that the mechanism through which the SNARE protein Syntaxin3 affected HCMV morphogenesis involved late endosomes/lysosomes since Syntaxin3 depletion reduced the expression of lysosomal membrane glycoproteins (LAMPs). We are now investigating how Syntaxin3 regulates LAMPs expression and the biological relevance of its depletion using as a model system the formation and secretion of lethal exosomes in T lymphocytes. A system that we have recently used to demonstrated a role for the diacylglycerol kinase alpha in the formation and polarisation of lysosomes-related organelles containing Fas ligand.

Herpes and poxviruses encode proteins with features similar to chemokine receptors, some of which have been implicated in viral pathogenesis. We have studied the cell biology of the chemokine receptors-like proteins encoded by HCMV. Of these virally-encoded chemokine receptors, US28 has been the most widely characterized. In collaboration with Drs Sergio Lira (NY, USA) and Martine Smit (Amsterdam, The Netherlands) we have found that expression of US28 in transgenic mice induces intestinal neoplasia and we have analysed the molecular mechanisms that underlay US28 proliferative signalling. We are now screening human samples from glioblastomas for the expression of HCMV chemokine receptors-like proteins in order to further support a role for HCMV in “oncomodulation”, the capacity of HCMV to infect tumor cells and increase their malignant properties.

Our research of the HCMV interactions with cellular membranes systems will provide crucial new insights to the cellular basis of herpesvirus replication and, in addition, shed light on basic aspects of cellular membrane biology. Furthermore, our studies on HCMV-encoded chemokine receptor like proteins will be important for understanding viral pathogenesis.

Our work is currently supported by grants from the Spanish Ministry of Economy and Competitiveness (PGC2018-098701-B-I00)

Previous grants:

Grupo Santander - Universidad Complutense de Madrid (PR41/17-20974)

Research Support Foundation of the State of Rio de Janeiro (E26/110.322/2014)

Programa PRONAMETRO of the the Brazilian Ministry of Development Industry and Foreign Trade

Complutense del Amo Fellowship

Spanish Ministry of Science and Technology (BFU2006-14379/BMC, BFU2009-07569/BMC and BFU20012-35067)

Comunidad de Madrid / CSIC (200620M034)