Attenuating mutations of the matrix gene of influenza A/WSN/33 virus

Abstract

The matrix protein (M1) of influenza virus plays an essential role in viral replication. Our previous studies have shown that basic amino acids 101RKLKR105 of M1 are involved in RNP binding and nuclear localization. For the present work, the functions of 101RKLKR105 were studied by introducing mutations into the M gene of influenza virus A/WSN/33 by reverse genetic methods. Individual substitution, R101S or R105S, had a minimal effect on viral replication. In contrast, the double mutation R101S-R105S was synergistic and resulted in temperature sensitivity reflected by reduced viral replication at a restrictive temperature. To investigate the in vivo effect on infection, BALB/c mice were infected with either A/WSN/33 wild-type (Wt) or mutant viruses and assessed for signs of illness, viral replication in the lungs, and survival rates. The results from mouse studies indicated that the R101S-R105S double mutant virus was strongly attenuated, while single mutant viruses R101S and R105S were minimally attenuated compared to A/WSN33 Wt under the same conditions. In challenge studies, mice immunized by infection with R101S-R105S were fully protected from lethal challenge with A/WSN/33. The replication and attenuating properties of R101S-R105S suggest its potential in development of live influenza virus vaccines.

FIG. 1.

(A) HA titers of the output mutant A/WSN/33 viruses in the initial culture fluids of the transfected cells. 293T/MDCK cells were transfected with a modified reverse genetic system to rescue M gene mutant viruses. The titers of viruses in supernatant of the transfected cells were measured by HAU, in the course of 48 to 144 h posttransfection. (B) Growth curves of purified mutant A/WSN/33 viruses in the culture fluids of virus-infected MDCK cells. The mutant viruses in panel A were purified by plaque plating on MDCK cells and amplified in the allantoic cavities. Confluent cell monolayers in 12-well plates were infected with A/WSN/33 Wt or with the mutant A/WSN/33 viruses at a multiplicity of infection of 0.001 at 37°C. At the indicated time points, infectious particles present in the culture fluids were titrated by plaque assay in MDCK cells.

FIG. 2.

Temperature sensitivity of M gene mutant viruses. The temperature-sensitive phenotype (ts) of the recombinant viruses was determined by plaque assay on MDCK cells at 33, 37, and 39.5°C. MDCK cells in 12-well plates were infected with 200 μl of 10-fold serially diluted virus and adsorbed at 37°C for 60 min. The inocula were removed and replaced with 1.5 ml of Eagle's minimal essential medium containing 0.7% agarose and 1 μg of TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone)-trypsin/ml. The infected cells were incubated at 33, 37, or 39.5°C. After 3 days' incubation, the cells were fixed with 100% methanol and stained with 0.1% crystal violet. The titer reduction at 39 to 33°C (A) or 37.5 to 33°C (B) was compared to that of Wt.

FIG. 3.

Percent loss of body weight of mice infected with M mutant viruses. Four-week-old BALB/c mice (six mice/group) were infected under isoflurane anesthesia with 5 × 10⁴ PFU of Wt or M gene mutant viruses/50 μl. Mice and body weight were observed and measured for 14 days. The body weights were compared with the body weights on day 0 after infection with viruses.

FIG. 4.

Survival rate of mice infected with M mutant viruses. Four-week-old BALB/c mice (six mice/group) were infected under isoflurane anesthesia with 5 × 10⁴ PFU of Wt or M gene mutant viruses/50 μl. Mice were monitored for 14 days.

FIG. 5.

Protection of mice infected with mutant influenza viruses and challenged with a lethal dose of Wt virus. Mice (six/group) were immunized intranasally with 5 × 10⁵, 5 × 10⁴, 5 × 10³, or 5 × 10² (5.7, 4.7, 3.7, and 2.7 log₁₀, respectively) PFU of R101S-R105S virus/50 μl. Animals receiving PBS served as a positive control. Two weeks after immunization, mice were challenged under isoflurane anesthesia with 5 × 10⁵ PFU of Wt virus/50 μl. The virus-challenged mice were monitored for body weight.