Image is here Professor

Department of Microbiology and Immunology
Center for Infectious Diseases
Ph.D., Temple University, 1984
Fellow American Academy of Microbiology
Fellow American Association for the Advancement of Scienc


Office:     (631) 632-7014
Fax:         (631) 632-7062


Viral Pathogenesis: Regulation of Endothelial Cell Responses

Viruses reprogram receptor and signaling responses in order to infect, spread and persist in host cells. To accomplish this viruses restrict antiviral pathways and dysregulate normal cellular functions that result in host, tissue and cell type specific disease. Endothelial cells (ECs) line the vasculature and maintain capillary barriers (Fig. 1). Our lab is primarily focused on viruses that infect ECs and the mechanisms by which infection of the endothelium directs pulmonary, neurologic and hemorrhagic disease. We address viral pathogenic mechanisms within primary human microvascular ECs using lentivirus KO and expression approaches. We analyze altered transcriptional and functional responses to reveal altered EC pathways and potential therapeutic targets to resolve disease.

Hantaviruses and Dengue viruses infect the endothelium and nonlytically cause hemorrhagic and edematous diseases by altering fluid barrier functions of the endothelium. Endothelial cells dynamically regulate fluid and immune cell emigration from capillaries while maintaining vascular integrity and vessel repair. In this setting failure is lethal and controlled by redundant fluid barrier failsafe mechanisms. Our lab is focused on defining mechanisms by which hantaviruses and dengue viruses regulate innate immune responses and induce vascular permeability responses following nonlytic infection of ECs.

Hemorrhagic and Edematous Disease: Pathogenic Mechanisms Pathogenic hantaviruses primarily infect ECs and cause two vascular leak syndromes, hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Pathogenic hantaviruses alter the function of endothelial cells β3 integrins, direct the recruitment of platelets to ECs (Fig. 2) and enhance the potent permeabilizing effects of VEGF on EC monolayers. Mechanisms by which hantaviruses ativate mTOR and hypoxia directed VEGF responses in primary human ECs are being investigated in order to define potential therapeutic targets that resolve EC barrier deficits. We found that hantaviruses directly enhance the permeability of ECs by activating RhoA, disrupting adherens junctions and exacerbating hypoxia directed permeability (Fig. 3).  Our findings are consistent with patient hypoxia and hypoxia directed VEGF induction that may contribute to the accumulation of up to 1 liter per hour of pulmonary edema fluid in HPS patients. Our recent findings indicate that a hantavirus protein binds directly to RhoGDI which normally keeps RhoA in an inactive state. These findings suggest targeting pathways that enhance RhoGDI interaction with RhoA and blocking RhoA directed permeability. 

While Dengue virus (DV) also causes hemorrhagic fever (DHF), mechanisms of DHF disease are distinct from hantavirus induced EC permeability. Consistent with DHF being an immune enhanced disease process, DV infection of ECs does not directly permeabilize ECs despite high levels of secreted NS1 protein that is a suggested cause of permeability.

We have demonstrated that primary human ECs are efficiently and productively infected (Fig. 4). We found that DV transiently infects ECs but does not establish a persistent infection and is cleared by induced IFN responses. The mechanism of DHF remains tied to infection of ECs and immune enhanced targeting of ECs following infection by a second serotype of DV.  Ultimately vascular permeability is the result of changes in the vascular endothelium and we have found that DV infected ECs secrete cytokines and chemokines that have the potential to contribute to immune enhanced pathogenesis. We are interested in determining the contribution of ECs to immune enhanced DV responses and the mechanism by which DV induces vascular permeability. These studies are aimed at defining viral and cellular targets for therapeutically regulating DV responses that contribute to the severity of disease in dengue patients.

Zika virus (ZIKV) and Powassan virus (POWV) infect brain microvascular ECs and cause neurologic damage by crossing the highly restrictive blood-brain-barrier (BBB) comprised of ECs, astrocytes and pericytes. ZIKV and POWV infections are respectively transmitted by mosquitoes and ticks, and cause severe neurologic diseases (ZIKV microcephaly; and highly lethal POWV encephalitis).  ZIKVs are unique flaviviruses that persist in patients for up to 6 months, are transmitted across the placenta and are sexually transmitted. We found that ZIKV and POWV persistently and nonlytically infect primary human brain microvascular ECs (Fig. 5) and this permits viral spread across the BBB and the lytic infection of neurons. ZIKV persistence requires regulation of the interferon (IFN) responses that otherwise block viral spread and replication in ECs. However, the mechanism by which ZIKV and POWV persistently infect ECs remains an enigma tightly linked to pathogenesis.

We found that ZIKV induces IFNβ/λ in ECs, but that ECs fail to produce IFNs and ZIKV selectively blocks the translation of IFN transcripts in ZIKV infected ECs. Our work focuses on studying how ZIKV targets unique destabilizing elements within the 3’UTR of IFN transcripts to prevent their expression. We also found that the ZIKV NS5 protein uniquely inhibits nuclear STAT1/2 and PML protein interactions that regulate IFN signaling. We established that SUMOylation of ZIKV NS5 protein regulates IFN responses in the nucleus of infected human brain ECs.

While IFNs are blocked, ZIKV induces and highly secretes chemokines from infected ECs that appear to be essential for the survival of ZIKV infected cells and ZIKV persistence in brain ECs. Both IFN regulation and ZIKV directed survival responses appear critical to ZIKV spread and persistence in ECs, allowing them to serve as reservoirs for viral spread systemically and across placental and blood brain barriers to cause neurological damage.

SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS)  characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on the regulation of EC responses. We found that the absence of ACE2 receptors on primary human ECs prevents SARS-CoV-2 from infecting pulmonary, cardiac, brain or renal ECs. However, when ECs are transduced to express recombinant ACE2 receptors, SARS-CoV-2 productively infects ECs resulting in high viral titers, multinucleate syncytia (Fig. 6) and cell lysis. ACE2-expressing ECs respond to SARS-CoV-2 infection by eliciting procoagulative and inflammatory responses. These findings indicate that SARS-CoV-2 indirectly triggers EC responses that regulate thrombosis and endotheliitis in COVID-19, and suggest therapeutically targeting epithelial and inflammatory responses that activate the endothelium. Our SARS-COV-2 studies are focused on defining epithelial, pericyte, and endothelial interactions that inhibit ACE2 function and activate endothelial RAS, BK, coagulation and inflammatory signals (Fig. 7).

Regulation of Innate Immunity Pathogenic viruses inhibit the early induction of IFN in order to replicate within ECs and we are defining virulence determinants and mechanisms used by hantavirus, DV, ZIKV and POWV proteins to regulate signaling pathways, IFNAR receptor responses and JAK/STAT responses that permit viral replication and persistence. These IFN regulating mechanisms have the potential to be therapeutically targeted to prevent persistence, permeability and viral crossing the BBB.

Hantavirus Therapeutics and Attenuation The hantavirus polymerase contains an N-terminal endonuclease domain that cleaves caps from cellular mRNAs to prime viral mRNA transcription. Hantaviruses uniquely snatch caps in the cytoplasm to prime transcription. This maintains a low level of replication required for viral persistence in ECs. We are defining the hantavirus endonuclease targeting mechanism that limits host cell protein shut off and polymerase activity to permit viral persistence. We are also defining cellular proteins required for cytoplasmic hantavirus cap snatching.

A distinct influenza virus endonuclease shuts down host cell protein synthesis and a small molecular inhibitor, baloxavir marboxyl, that blocks endonuclease activity is an influenza therapeutic. As influenza and hantavirus endonucleases share binding pocket structures, we are evaluating baloxavir derivatives for their ability to inhibit the hantavirus endonuclease and serve as hantavirus therapeutics. Reverse genetic approaches are being developed in order to attenuate pathogenic hantaviruses.



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Lab Members

Elena Gorbunova, Ph.D., Senior Research Associate

Megan Mladinich, M.S., Postdoctoral Fellow - IRACDA Scholar

Grace Himmler, B.S., Graduate Student - Molecular and Cell Biology PhD Program

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Selected Publications

Conde, J.N., Schutt, W., Gorbunova, E.E., Mackow, E.R. Recombinant ACE2 Expression is Required for SARS-CoV-2 to Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses. mBio 11/2020 in press.

Conde, J.N., Schutt, W., Mladinich, M., Sohn, S.Y., Hearing, P., Mackow, E.R. 2020. NS5 Sumoylation Directs Nuclear Responses that Permit Zika Virus to Persistently Infect Human Brain Microvascular Endothelial Cells. J Virology doi:10.1128/JVI.01086-20.

Simons, M.J., Gorbunova, E.E., Mackow, E.R. 2019. Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386. J. Virology 93:e00338-19.

Mladinich, M., Schwedes, J., Mackow, E.R. 2017. Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells. mBio 8, 4 e00952-17.

Gorbunova, E. Simons, M. Gavrilovskaya, I, Mackow, E.R. 2016. The Andes Virus NP Directs Basal Endothelial Cell Permeability by Activating RhoA. mBio 7(5):e01747-16.

Mackow, E.R., Gorbunova, E. Gavrilovskaya, I.N. 2015. Endothelial cell dysfunction in pathogen-induced hemorrhagic fevers. Frontiers in Microbiology.

Dalrymple, N., Cimica, V. and Mackow, E.R. 2015. Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant.  mBio 6:e00553-00515.

Cimica, V, Dalrymple, E. Roth, A. Nasonov, E. Mackow. 2014. An Innate Immunity Regulating Virulence Determinant is Uniquely Encoded within the Andes Virus Nucleocapsid Protein. mBio  5(1):e01088-13.

Matthys, V, V. Cimica, N. Dalrymple, N. Glennon, C. Bianco, Mackow, E.R.. 2014. Hantavirus GnT Elements Mediate TRAF3 Binding and Inhibit RIG-I/TBK1 directed IFN Transcription by Blocking IRF3 Phosphorylation. Journal of Virology. 88: 2246-2259.

Dalrymple, N., and Mackow, E.R. 2012. Endothelial Cells Elicit Immune Enhancing Responses to Dengue Virus Infection. Journal of Virology 86 (12) 6408-15.

Gavrilovskaya, I. N., Gorbunova, E. , Mackow, N.A., Mackow, E.R. 2008. Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability. J. Virology 82:5797-806.

Raymond, T., Gorbunova, E., Gavrilovskaya, I.N., Mackow, E.R. 2005. Pathogenic Hantaviruses bind plexin semaphorin- integrin domains present at the apex of inactive, bent αvβ3 integrin conformers. Proc. Nat. Acad. Sci. USA 102, p1163-8.

Gavrilovskaya, I.N., Shepley, M., Shaw, R. D., Ginsberg, M.H, Mackow, E.R. 1998. β3 Integrins Mediate the Cellular Entry of Hantaviruses that Cause Respiratory Failure. Proc. Nat .Acad. Sci. 95: 7074-7079.