The respiratory syncytial virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly.

The M2-2 protein from the respiratory syncytial virus (RSV) is a 10 kDa protein expressed by the second ORF of the viral gene M2. During infection, M2-2 has been described as the polymerase cofactor responsible for promoting genome replication, which occurs by the induction of changes in interactions between the polymerase and other viral proteins at early stages of infection. Despite its well-explored role in the regulation of the polymerase activity, little has been made to investigate the relationship of M2-2 with cellular proteins. A previous report showed poor recruitment of M2-2 to viral structures, with the protein being mainly localized to the nucleus and cytoplasmic granules. To unravel which other functions M2-2 exerts during infection, we performed proteomic analysis of co-immunoprecipitated cellular partners, identifying enrichment of proteins involved with regulation of translation, protein folding and mRNA splicing. In approaches based on these data, we found that M2-2 expression downregulates eiF2α phosphorylation and inhibits both translation and stress granules assembly. Finally, we also verified that M2-2 is targeted for proteasome degradation, being localized to granules composed of defective ribosomal products at the cytoplasm. These results suggest that besides its functions in the replicative complex, M2-2 may exert additional functions to contribute to successful RSV infection.

Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target.

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.

Molecular mechanisms and pharmacological interventions in the replication cycle of human coronaviruses.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), as well as SARS-CoV from 2003 along with MERS-CoV from 2012, is a member of the Betacoronavirus genus of the Nidovirales order and is currently the cause of the pandemic called COVID-19 (or Coronavirus disease 2019). COVID-19, which is characterized by cough, fever, fatigue, and severe cases of pneumonia, has affected more than 23 million people worldwide until August 25th, 2020. Here, we present a review of the cellular mechanisms associated with human coronavirus replication, including the unique molecular events related to the replication transcription complex (RTC) of coronaviruses. We also present information regarding the interactions between each viral protein and cellular proteins associated to known host-pathogen implications for the coronavirus biology. Finally, a specific topic addresses the current attempts for pharmacological interventions against COVID-19, highlighting the possible effects of each drug on the molecular events of viral replication. This review intends to aid future studies for a better understanding of the SARS-CoV-2 replication cycle and the development of pharmacological approaches targeting COVID-19.

Human respiratory syncytial virus detection in children admitted at a community hospital in Botucatu, SP, Brazil

O Vírus Respiratório Sincicial Humano (VRSH) é descrito como o mais importante patógeno viral causador de doenças respiratórias agudas das vias respiratórias inferiores em crianças. Neste estudo 84 amostras de crianças com idade abaixo dos dois anos apresentando sintomas de doença respiratória aguda, foram obtidas no período de setembro de 2000 a novembro de 2001. Analise por imunofluorescência indireta e transcrição reversa seguida de PCR, revelou que 18 per center (15/84) das amostras foram positivas, sendo que em 80 per center (12/15) dos casos a detecção de VRSH foi observada em crianças abaixo dos seis meses, e também que os subgrupos A e B co-circularam. Estes são os primeiros dados obtidos para a cidade de Botucatu, sendo que a sazonalidade mostrou-se evidente pela maior circulação desse vírus entre os meses de maio e julho.

Construction of adenoviral vectors expressing F and G glycoproteins of human respiratory syncytial virus (HRSV)

O Vírus Sincicial Respiratório Humano (HRSV) foi isolado e caracterizado pela primeira vez em 1957 e é considerado como o patógeno viral mais freqüente do trato respiratório de bebês e crianças. Apesar de muitos anos de pesquisa, não há ainda um tratamento específico ou uma vacina licenciada. Seu genoma é composto por uma fita simples de RNA polaridade negativa e o vírion consiste em um nucleocapsídeo empacotado por um envelope lipídico. O envelope contém projeções, chamadas espículas, constituídas de homoligômeros de uma das 3 glicoproteínas de membrana: a proteína de ligação G ("attachment"), a proteína de fusão F ("fusion") e a proteína SH ("small hydrofobic"). O objetivo deste trabalho foi construir dois adenovirus recombinantes defectivos em replicação expressando separadamente os genes F e G do HRSV. Este sistema foi escolhido porque os vetores adenovirais possuem a capacidade de inserir genes em uma grande variedade de linhagens celulares in vitro e in vivo. Para obtenção destes vetores adenovirais, um RT-PCR de RNA extraído do protótipo A2 de HRSV foi feito e os genes F e G clonados em vetores pAdeno-X. pAdeno-F e pAdeno-G foram transfectados em células HEK-293 para a produção do vírus recombinante, que expressaram corretamente essas duas proteínas constituem-se ferramentas para imunização e estudos funcionais.

Complementation of the DNA repair deficiency in human xeroderma pigmentosum group a and C cells by recombinant adenovirus-mediated gene transfer.

Nucleotide excision repair (NER) is one of the most versatile DNA repair mechanisms, ensuring the proper functioning and trustworthy transmission of genetic information in all living cells. The phenotypic consequences caused by NER defects in humans are autosomal recessive diseases such as xeroderma pigmentosum (XP). This syndrome is the most sun-sensitive disorder leading to a high frequency of skin cancer. The majority of patients with XP carry mutations in the XPA or XPC genes that encode proteins involved in recognition of DNA damage induced by UV light at the beginning of the NER process. Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair. So far there is no effective long-term treatment for these patients. Skin cancer prevention can only be achieved by strict avoidance of sunlight exposure or by the use of sunscreen agents. We have constructed recombinant adenoviruses carrying the XPA and XPC genes that were used to infect XP-A and XP-C immortalized and primary fibroblast cell lines. UV survival curves and unscheduled DNA synthesis confirmed complete phenotypic reversion in XP DNA repair deficient cells with no trace of cytotoxicity. Moreover, transgene expression is stable for at least 60 days after infection. This efficient adenovirus gene delivery approach may be an important tool to better understand XP deficiency and the causes of DNA damage induced skin cancer.