Tula orthohantavirus nucleocapsid protein is cleaved in infected cells and may sequester activated caspase-3 during persistent infection to suppress apoptosis.

The family Hantaviridae mostly comprises rodent-borne segmented negative-sense RNA viruses, many of which are capable of causing devastating disease in humans. In contrast, hantavirus infection of rodent hosts results in a persistent and inapparent infection through their ability to evade immune detection and inhibit apoptosis. In this study, we used Tula hantavirus (TULV) to investigate the interplay between viral and host apoptotic responses during early, peak and persistent phases of virus infection in cell culture. Examination of early-phase TULV infection revealed that infected cells were refractory to apoptosis, as evidenced by the complete lack of cleaved caspase-3 (casp-3C) staining, whereas in non-infected bystander cells casp-3C was highly abundant. Interestingly, at later time points, casp-3C was abundant in infected cells, but the cells remained viable and able to continue shedding infectious virus, and together these observations were suggestive of a TULV-associated apoptotic block. To investigate this block, we viewed TULV-infected cells using laser scanning confocal and wide-field deconvolution microscopy, which revealed that TULV nucleocapsid protein (NP) colocalized with, and sequestered, casp-3C within cytoplasmic ultrastructures. Consistent with casp-3C colocalization, we showed for the first time that TULV NP was cleaved in cells and that TULV NP and casp-3C could be co-immunoprecipitated, suggesting that this interaction was stable and thus unlikely to be solely confined to NP binding as a substrate to the casp-3C active site. To account for these findings, we propose a novel mechanism by which TULV NP inhibits apoptosis by spatially sequestering casp-3C from its downstream apoptotic targets within the cytosol.

Macropinocytosis contributes to hantavirus entry into human airway epithelial cells.

Hantaviruses are emerging rodent-borne negative-strand RNA viruses associated with severe human diseases. Zoonotic transmission occurs via aerosols of contaminated rodent excreta and cells of the human respiratory epithelium represent likely early targets. Here we investigated cellular factors involved in entry of the pathogenic Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV) into human respiratory epithelial cells. Screening of a kinase inhibitor library using a biocontained recombinant vesicular stomatitis virus pseudotype platform revealed differential requirement for host kinases for HTNV and ANDV entry and provided first hints for an involvement of macropinocytosis. Examination of a selected panel of well-defined inhibitors of endocytosis confirmed that both HTNV and ANDV enter human respiratory epithelial cells via a pathway that critically depends on sodium proton exchangers and actin, hallmarks of macropinocytosis. However, HTNV and ANDV differed in their individual requirements for regulatory factors of macropinocytosis, indicating virus-specific differences.

Serum levels of metalloproteinases and their inhibitors during infection with pathogens having integrin receptor-mediated cellular entry.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with numerous roles in the normal immune response to infection. However, excess MMP activity following infection may lead to immunopathological processes that cause tissue damage. Their activity in normal tissues is subject to tight control, which is regulated by its specific endogenous tissue inhibitors (TIMPs). It is known that MMPs bind to cell surface proteins (e.g. integrins) and that such interactions can have modulatory effects on MMP functionality. The objective of this study was to determine whether there are differences in MMP and TIMP production during the acute phase of infection with different pathogens that use ß-integrins as their receptors for cell entry. METHODS: We measured the total amounts of soluble MMP-2, MMP-9, TIMP-1, and TIMP-2 in the sera from patients infected with Dobrava virus (DOBV), Coxiella burnetii, or uropathogenic Escherichia coli. Statistical analyses were used to correlate MMP/TIMP serum levels with different clinical laboratory parameters. RESULTS: The results showed that both of the bacterial infections generally manifested the stronger effect on MMP production, while in contrast, viral infection introduced stronger changes to metalloproteinase inhibitors. MMPs and TIMPs were significantly correlated with some of the clinical laboratory parameters in both bacterial infections, but no correlations were found for DOBV infection. CONCLUSIONS: These findings suggest diverse mechanisms by which MMP activity could be implicated in the pathology of these 2 bacterial infections versus the viral DOBV infection, despite the type of their cellular entry receptors.

Prevalence of anti-hantavirus antibodies in patients with hypertransaminemia in Madrid (Spain).

The hantaviruses are involved in a number of clinical syndromes of different severity and prognosis. Hantaviruses are widely distributed around the world, but the spectrum of illnesses they cause outside recognized endemic areas is unclear. A retrospective analysis was performed to detect anti-hantavirus antibodies in the serum of patients with hypertransaminemia of unknown etiology and in that of healthy members of the general population of Madrid (Spain). Antibodies were detected by indirect immunofluorescence and enzyme immunoassay; positive results were confirmed by Western blotting. Of the 182 patients with hypertransaminemia, 11 (6%) were positive for anti-hantavirus IgG antibodies; Western blotting using recombinant Puumala virus N antigen showed one of these patients to have hantavirus-specific IgM antibodies. Among the 146 healthy subjects from the general population, 3 (2%) were positive for anti-hantavirus IgG antibodies. These results show that anti-hantavirus antibodies are more commonly detected in patients with hypertransaminemia than in healthy people.