Reduced levels of neuraminidase of influenza A viruses correlate with attenuated phenotypes in mice

Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3-and 5-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3 and 5 NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3-and 5-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3 and 5 NTRs to associate through basepairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3 and 5 NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3 and 5 NTRs were selected.

Section: Discussionmentioning

confidence: 99%

Bunyamwera Bunyavirus RNA Synthesis Requires Cooperation of 3′- and 5′-Terminal Sequences

Barr

Wertz

2004

“…1) (5,13,19,35). The same mutation was found to be responsible for a reduction in the levels of mRNA and protein expression when introduced into the NA, PA, and NS segments of recombinant viruses (5,14,46). For this purpose, the M RNA segment was PCR amplified using as template pPOLI-M plasmid (13) and primers 5Ј-GATCGCTCTTCGGCCAGCGAAAGCATGTAG ATATTGAAAGATG-3Ј (positive sense; M1/M2 start codon is underlined; inserted promoter mutation is in boldface) and 5Ј-GATCGCTCTTCTATTAGTAGAAACAATGTAGTTTT TTACTCC-3Ј (negative sense; M2 stop codon is underlined; inserted promoter mutation is in boldface).…”

mentioning

confidence: 92%

The Morphology and Composition of Influenza A Virus Particles Are Not Affected by Low Levels of M1 and M2 Proteins in Infected Cells

Bourmakina

Garcı́a-Sastre

2005

We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.Influenza A virus is an enveloped virus of the Orthomyxoviridae family with a segmented negative-strand RNA genome. Its eight viral RNA (vRNA) segments encode the three subunits of the viral RNA polymerase complex, PB1, PB2, and PA; the nucleoprotein NP; the glycoproteins HA and NA; the matrix protein M1; the ion channel protein M2; the nonstructural protein NS1; the nuclear export protein NEP; and the recently discovered facilitator of apoptosis PB1-F2 (7). The M1 protein is a major structural component of the virus particles and forms a layer underneath the lipid cell-derived envelope (43,44). Inside the virions and in infected cells at late stages of the virus replication, the M1 protein associates with the viral ribonucleoproteins (vRNPs), which are composed of viral RNA molecules, multiple copies of the NP, and the three subunits of the viral polymerase holding the ends of the viral RNAs (3, 41, 51). Transcription and replication of the influenza virus genome take place in the nucleus of infected cells. By contrast, assembly of virus particles occurs at the apical surface of the plasma membrane where the M1 protein presumably interacts on one side with the cytoplasmic tails of the glycoproteins anchored in the membrane and on the other side with the vRNPs. Although a direct interaction of the M1 protein with the viral glycoproteins was not demonstrated, expression of HA and NA proteins stimulates membrane association of M1 protein (11). Moreover, a mutant virus with deleted cytoplasmic tails at both HA and NA proteins shows severe defects in particle morphology, genome packaging, and incorporation of NA and M1 into virions (25,55).Previous works show that the M1 protein plays a pivotal role in influenza A virus budding. Mutation of certain residues in the M1 protein dramatically affects the morphology of the virus particles (4, 9

“…Furthermore, many mutations in the influenza viral promoter result in nonviable isolates, as evidenced by the difficulty of generating such mutants. Thus, extending the in vitro reporter-based findings to an in vivo setting is limited, and only a few recombinant viruses bearing promoter mutations have been generated (2,3,12,28).…”

mentioning

confidence: 99%

Recombinant Influenza A Viruses with Enhanced Levels of PB1 and PA Viral Protein Expression

Belicha-Villanueva

Rodríguez-Madoz

Maamary

et al. 2012

Influenza A viruses containing the promoter mutations G3A/C8U in a given segment express increased levels of the corresponding viral protein during infection due to increased levels of mRNA or cRNA species. The replication of these recombinant viruses is attenuated, and they have an enhanced shedding of noninfectious particles and are incapable of antagonizing interferon (IFN) effectively. Our findings highlight the possibility of increasing influenza virus protein expression and the need for a delicate balance between influenza viral replication, protein expression, and assembly.