The M1 and NP proteins of influenza A virus form homo- but not heterooligomeric complexes when coexpressed in BHK-21 cells.

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (⌬YRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/ K102A, ⌬YRKL, 4A, PTAP, 4A؉PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.Influenza virus matrix protein (M1) is a relatively small, highly conserved protein (252 amino acids [aa] in type A and 248 aa in type B viruses). M1 is the most abundant protein in virus particle and plays critical roles in many aspects of virus replication. These include (i) dissociation of M1 from the M1/ viral ribonucleoprotein (vRNP) complex during the entry and uncoating of infecting virus, (ii) nuclear entry of M1, (iii) interaction of M1 with vRNP to form M1/vRNP complex, (iv) role of M1 in the exit of vRNP from the nucleus into the cytoplasm, (v) interaction of M1 with viral envelope proteins (hemagglutinin [HA], neuraminidase, and ion channel M2), (vi) membrane binding of M1, (vii) dimer/oligomer formation of M1, (viii) role of M1 in virus budding, including recruitment of viral components at the assembly site and recruitment of host components for budding and release of virus particles (reviewed in references 29, 40, and 41).The M1 monomer is 60 Å long (55), possessing two globular regions (aa 1 to 164 and 165 to 252) linked by a proteasesensitive loop. The structure mostly consist...

mentioning

confidence: 99%

Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

Hui¹,

Barman²,

Yang³

et al. 2003

“…In contrast, numerous reports in the literature have documented that the multimerization of matrix (tegument) proteins in structurally less complex RNA viruses play an essential role in virus assembly, particularly in the envelopment (budding) of the particle. As examples, the MAs in vesicular stomatitis virus, the paramyxovirus SV5, Ebola virus, influenza virus, Rous sarcoma virus, and human immunodeficiency virus type 1 (HIV-1) are membrane associated and are proposed to undergo multimerization to create lattice-like structures that have been proposed to lead to the eventual budding or pinching off of that region of the membrane (11,13,15,16,18,20,21,27,33,35,38,39,47,54). Although the recruitment of host cellular functions is necessary for the budding of these viruses, multimerization of their MAs represents an essential viral function in this process (17,19,28,33).…”

Section: Discussionmentioning

confidence: 99%

Multimerization of Tegument Protein pp28 within the Assembly Compartment Is Required for Cytoplasmic Envelopment of Human Cytomegalovirus

Seo

Britt

2008

Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.Human cytomegalovirus (HCMV) is an important human pathogen associated with acute and chronic disease in both normal and immunocompromised populations (4,34,45,46). The virus is the largest and most complex member of the family of human herpesviruses. The virion of HCMV consists of three distinct structures: a nucleocapsid containing a 230-kbp linear DNA genome that may encode over 200 open reading frames (ORFs), an envelope including an as-yet-undefined number of viral glycoproteins, and a tegument layer located between the capsid and envelope (31,32,49,53,55). In addition, the virion contains a minimum of 71 virus-encoded proteins and a large number of host-derived proteins (50).HCMV assembly is a multistage, poorly understood process. Although all proposed models include well-studied mechanisms of capsid assembly within the nucleus of infected cells, the final tegumentation and envelopment in the cytoplasm of infected cells remains poorly understood (29). Early ultrastructural studies of herpes simplex virus (HSV) and HCMV noted that nonenveloped cytoplasmic particles in HCMV-infected cells were coated with a thick tegument layer but that nonenveloped HSV particles often had the appearance of naked capsids (44). These and other findings suggested that, in contrast to the assembly of HSV and other alphaherpesviruses, tegument proteins could play a key role in the cytoplasmic phase of HCMV assembly and that the cytoplasmic assembly of HSV and...

“…It is known that the M1 protein forms homo-oligomers when expressed from a cDNA (54). Moreover, the three-dimensional structure of an amino-terminal fragment of the M1 protein predicts that this protein can form dimers which could self-assemble into large polymers (45).…”

Section: M1 As the Key Element In Influenza Virus Particle Formationmentioning

confidence: 99%

“…COS-1-transfected cultures were harvested and fractionated as described by Zhao et al (54). Briefly, 3 ϫ 10 6 cells were rinsed with phosphate-buffered saline and scraped into 1 ml of a buffer containing 10% (wt/vol) sucrose and 10 mM Tris-HCl (pH 7.5).…”

mentioning

confidence: 99%

Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding

Gómez‐Puertas¹,

Albó²,

Pérez-Pastrana

et al. 2000

240

238

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.The final step in the lytic cycle of enveloped viruses involves the budding of the newly formed particles from cellular membranes. Previous to this step, all viral structural components should have been transported, either individually or as preassembled complexes, to the cellular membrane, where viral proteins will drive the budding process.A number of studies have focused on the assembly and budding processes of viruses (arena-, alpha-, rhabdo-, paramyxo-, orthomyxo-, and retroviruses) that obtain their envelope from the plasma membrane (reviewed in references 2, 13, and 19). For the alphavirus Semliki Forest virus, it has been established that virus budding is strictly dependent on interactions between the transmembrane spike protein and the internal nucleocapsid (46). In retroviruses, however, interactions between the cytoplasmic tail of external virus proteins (Env) and the internal virus components (Gag polyprotein) are not a prerequisite for virus budding since expression of the Gag protein alone is sufficient to drive budding of virus-like particles (VLPs) (7,14). A different mechanism, which directs the assembly and release of coronavirus particles, which assemble at intracellular membranes, has been described (47). In this case, expression of viral membrane proteins alone is sufficient to drive the assembly and budding of VLPs (47).It is widely accepted that the matrix protein plays a pivotal role as an assembly organizer for RNA viruses containing a single negative-strand genomic RNA molecule (such as rhabdo-and paramyxoviruses) (reviewed in reference 25). In fact, rabies and measles viruses modified by reverse genetics technology to lack the matrix gene grow poorly, and the released matrix-less particles show drastically altered morphologies (3, 31). Moreover, it has been shown that the M1 proteins of vesicular stomatits virus (VSV) and human parainfluenza virus type 1 have intrinsic budding activity when expressed alone (5, 22, 26), an observation ...